Final Report Summary - PEG-ANION-NET (Proteomics & Epi-Genetics of Anion-transport-Network)
The aim of the exchange programme is to fortify and intensify the scientific exchange and connectivity between four groups - two in Europe (European-Consortium) and two in the USA - that have a proven record in collaboration over the past. The common theme, on which the scientific collaborations are based, is “Proteomics & Epi-Genetics of Anion transport” (PEG-Anion). Epi-Genetics within this collaboration refers to how inherited genetic information, and genetic information not encoded in the DNA sequence translates into variations in overall function of the gene products. Extending the existing collaboration between the PEG-Anion partner-laboratories into a PEG-ANION-NETWORK (PEG-ANION-NET) significantly increased the momentum to the knowledge-transfer between the four groups. The groups have individual funding for the respective PEG-Anion projects, and the research exchange scheme (IRSES) helped to successfully strengthen, fortify, and ultimately sustain the research activity between the four groups beyond the actual projects by covering the mobility expenses induced by the planned PEG-ANION-NET activity.
The sequencing of the human genome was a milestone in science and marked the beginning of the post-genomic era. The knowledge of the genomic make up, however, does not by itself give much insight into the function of single genes, nor does it give conclusive answers on the effect of single genes or gene products in regulatory circuits established in living cells. The challenge now and in the immediate future is to combine the genomic- and epigenetic-information with functional-information of the gene products in their native environment. The research in the PEG-Anion partner-laboratories is focused on the proteomics, genomics and epigenetics of two anion ‘transporters’ and how the proteomic, genomic and epigenetic information translates into the respective gene product function. Genetic make-up, proteomic interaction and epigenetic regulation of gene expression are important for normal cellular physiology and play key roles in both disease progression and responses to therapeutic intervention.
The expertise and research activities within the PEG-Anion partners cover a broad range of topics. Within the scope of the proposal it is impossible to include all fields in which the four partners are active. In the past, the work on two anion-transporters tied together the research interest of the four groups. The two anion-transporters under investigation are (i) ICln, and (ii) Pendrin.
ICln: ICln was first cloned from Madin Darby Canine Kidney (MDCK) cells using expression cloning in Xenopus laevis oocytes (Paulmichl et al., Nature, 1992, 356:238-241). Reconstitution of ICln in lipid-bilayers leads to ion channels, whose selectivity depends on the lipid environment. Expression of ICln in Xenopus oocytes resulted in a nucleotide-sensitive, outwardly rectifying Cl- current that closely resembled the biophysical characteristics of the swelling dependent anion and osmolyte channel (IClswell), suggesting that ICln is a molecular candidate for IClswell. Our experimental results and the reports from various other laboratories functionally linked ICln to cell volume regulation and IClswell. For instance, in the kidney, ICln is located in the apical membrane of proximal tubule cells, as well as in the membranes and cytosol of distal tubule cells. This distribution pattern can be altered by furosemide-stimulated diuresis, which induces hypotonic stress. Similarly, ICln translocates from the cytosol to the cell membrane under hypotonic conditions in a variety of continuous cell-lines. Furthermore, in heart cells, ICln translocation by hypotonicity is paralleled by taurine efflux. As expected, over-expression of ICln in mammalian cells leads to accelerated activation and increased current amplitudes of IClswell during a hypotonic challenge, while a functional knock-down of ICln shows the opposite effect on IClswell activation and current density. Another major focus of our work, besides the involvement of ICln in volume regulation, is the identification and characterization of other functional modules in which ICln is involved. ICln binds to a variety of cytosolic proteins like actin, the erythrocyte protein 4.1 the non-muscle isoform of the alkali myosin light chain, like-Sm (LSm) proteins, the Janus kinase-binding protein 1 (JBP1; also known as protein arginine methyltransferase 5 (PRMT5)), and the casein kinase type I or II (CK-I/CK-II) like kinase. Determining the interactome of ICln, i.e. the structure-function relationship between ICln and its binding partners, and characterizing their spatio-temporal organization is of utmost importance in order to understand the role of ICln in volume regulation and ultimately in human diseases.
Pendrin: The SLC26A4 gene codes for the pendrin protein (PD), which exchanges chloride, iodide and other anions for bicarbonate. Pendrin is mainly expressed in the thyroid gland, inner ear and kidney. The identification of the PD coding gene (SLC26A4 or PDS) allowed investigations on the origin of some types of hereditary deafness. 170 mutations located both in exons and introns of the PD gene have been linked to various human diseases. Individuals with disease causing mutations can present distinct phenotypes named Pendred syndrome (PS, OMIM n° 274600), DFNB4 (OMIM n° 600792) or LVAS (OMIM n° 603545). Pendred’s syndrome is a relatively common (1:10.000) autosomal recessive disorder characterized by sensorineural hearing loss, and is often associated with dyshormonogenic goitre. PS differs from other hereditary forms of early onset deafness in that its phenotype is variable with respect to the time of onset of deafness, or the presence and extent of vestibular alterations. Determining the functional implications of SLC26A4 mutations, as well as determining the functional role and epigenetic regulation of Pendrin, will help in the future to better assist patients with Pendred-syndrome.
Description of the Work Packages divided by specific tasks
The PEG-ANION-NET project consists of two work packages. With the first focusing on ICln (A) and the second focusing on Pendrin (B). Each work package is divided into the following specific tasks:
(A) (1) ICln interactome, (2) ICln function in the kidney, (3) ICln genetics, (4) ICln epigenetics, (5) ICln function in arrhythmias;
(B) (6) Pendrin genetics, (7) functional characterisation of Pendrin- mutants, (8) Pendrin epigenetics, and (9) Pendrin function in the kidney.
In the first two years we focused, according to our time table on tasks 1,2,4,5,6,7 and 8.
In the second two years we focused, in addition to the former, also on tsks 3 and 9.
In the first two years the research results where published in 5 original papers in the prestigious journals like Clinical Pharmacology and Therapeutics and Cell. Physiol. Biochemistry (see below).
In the second two years we published 13 additional original paper in prestigious journals as Nature Reviews Drug Discovery (2 articles), PLOS ONE and Cell. Physiol. Biochemistry (see below).
One manuscript is in preparation for Cell (will be submitted in 2014).
In addition, due to the momentum generated by the IRSES network we applied and successfully obtained the right to organize an 'Exploratory Workshop' sponsored by the ESF in 2011.
The follow-up meeting was organized in Lake Cumberland (USA) in 2013.
The scientific results obtained during the IRSES funding have been published again in a special issue of Cellular Physiology and Biochemistry in 2013.
The sequencing of the human genome was a milestone in science and marked the beginning of the post-genomic era. The knowledge of the genomic make up, however, does not by itself give much insight into the function of single genes, nor does it give conclusive answers on the effect of single genes or gene products in regulatory circuits established in living cells. The challenge now and in the immediate future is to combine the genomic- and epigenetic-information with functional-information of the gene products in their native environment. The research in the PEG-Anion partner-laboratories is focused on the proteomics, genomics and epigenetics of two anion ‘transporters’ and how the proteomic, genomic and epigenetic information translates into the respective gene product function. Genetic make-up, proteomic interaction and epigenetic regulation of gene expression are important for normal cellular physiology and play key roles in both disease progression and responses to therapeutic intervention.
The expertise and research activities within the PEG-Anion partners cover a broad range of topics. Within the scope of the proposal it is impossible to include all fields in which the four partners are active. In the past, the work on two anion-transporters tied together the research interest of the four groups. The two anion-transporters under investigation are (i) ICln, and (ii) Pendrin.
ICln: ICln was first cloned from Madin Darby Canine Kidney (MDCK) cells using expression cloning in Xenopus laevis oocytes (Paulmichl et al., Nature, 1992, 356:238-241). Reconstitution of ICln in lipid-bilayers leads to ion channels, whose selectivity depends on the lipid environment. Expression of ICln in Xenopus oocytes resulted in a nucleotide-sensitive, outwardly rectifying Cl- current that closely resembled the biophysical characteristics of the swelling dependent anion and osmolyte channel (IClswell), suggesting that ICln is a molecular candidate for IClswell. Our experimental results and the reports from various other laboratories functionally linked ICln to cell volume regulation and IClswell. For instance, in the kidney, ICln is located in the apical membrane of proximal tubule cells, as well as in the membranes and cytosol of distal tubule cells. This distribution pattern can be altered by furosemide-stimulated diuresis, which induces hypotonic stress. Similarly, ICln translocates from the cytosol to the cell membrane under hypotonic conditions in a variety of continuous cell-lines. Furthermore, in heart cells, ICln translocation by hypotonicity is paralleled by taurine efflux. As expected, over-expression of ICln in mammalian cells leads to accelerated activation and increased current amplitudes of IClswell during a hypotonic challenge, while a functional knock-down of ICln shows the opposite effect on IClswell activation and current density. Another major focus of our work, besides the involvement of ICln in volume regulation, is the identification and characterization of other functional modules in which ICln is involved. ICln binds to a variety of cytosolic proteins like actin, the erythrocyte protein 4.1 the non-muscle isoform of the alkali myosin light chain, like-Sm (LSm) proteins, the Janus kinase-binding protein 1 (JBP1; also known as protein arginine methyltransferase 5 (PRMT5)), and the casein kinase type I or II (CK-I/CK-II) like kinase. Determining the interactome of ICln, i.e. the structure-function relationship between ICln and its binding partners, and characterizing their spatio-temporal organization is of utmost importance in order to understand the role of ICln in volume regulation and ultimately in human diseases.
Pendrin: The SLC26A4 gene codes for the pendrin protein (PD), which exchanges chloride, iodide and other anions for bicarbonate. Pendrin is mainly expressed in the thyroid gland, inner ear and kidney. The identification of the PD coding gene (SLC26A4 or PDS) allowed investigations on the origin of some types of hereditary deafness. 170 mutations located both in exons and introns of the PD gene have been linked to various human diseases. Individuals with disease causing mutations can present distinct phenotypes named Pendred syndrome (PS, OMIM n° 274600), DFNB4 (OMIM n° 600792) or LVAS (OMIM n° 603545). Pendred’s syndrome is a relatively common (1:10.000) autosomal recessive disorder characterized by sensorineural hearing loss, and is often associated with dyshormonogenic goitre. PS differs from other hereditary forms of early onset deafness in that its phenotype is variable with respect to the time of onset of deafness, or the presence and extent of vestibular alterations. Determining the functional implications of SLC26A4 mutations, as well as determining the functional role and epigenetic regulation of Pendrin, will help in the future to better assist patients with Pendred-syndrome.
Description of the Work Packages divided by specific tasks
The PEG-ANION-NET project consists of two work packages. With the first focusing on ICln (A) and the second focusing on Pendrin (B). Each work package is divided into the following specific tasks:
(A) (1) ICln interactome, (2) ICln function in the kidney, (3) ICln genetics, (4) ICln epigenetics, (5) ICln function in arrhythmias;
(B) (6) Pendrin genetics, (7) functional characterisation of Pendrin- mutants, (8) Pendrin epigenetics, and (9) Pendrin function in the kidney.
In the first two years we focused, according to our time table on tasks 1,2,4,5,6,7 and 8.
In the second two years we focused, in addition to the former, also on tsks 3 and 9.
In the first two years the research results where published in 5 original papers in the prestigious journals like Clinical Pharmacology and Therapeutics and Cell. Physiol. Biochemistry (see below).
In the second two years we published 13 additional original paper in prestigious journals as Nature Reviews Drug Discovery (2 articles), PLOS ONE and Cell. Physiol. Biochemistry (see below).
One manuscript is in preparation for Cell (will be submitted in 2014).
In addition, due to the momentum generated by the IRSES network we applied and successfully obtained the right to organize an 'Exploratory Workshop' sponsored by the ESF in 2011.
The follow-up meeting was organized in Lake Cumberland (USA) in 2013.
The scientific results obtained during the IRSES funding have been published again in a special issue of Cellular Physiology and Biochemistry in 2013.