DISSERTATION ORGANIZATION
DISCUSSION
Our protein expression profiling identified 22 proteins that are differentially
expressed in RPCs when compared to BPCs isolated from genetically similar mice. Four of the differentially expressed proteins correspond to proteins known to be involved in a cellular response to stress, and analysis of potential transcription factor binding sites in the promoter regions of their genes suggests these proteins could be co-regulated at the transcriptional level.
Potential Developmental Role of Stress-Response Proteins Expressed by RPCs
Heat shock proteins (Hsp) are a group of proteins that are present in all cells at all biological levels. These proteins are needed for normal cell growth and maintenance and have been detected during embryogenesis in various organisms (Calabrese et al., 2002). Different developmental profiles of Hsps have suggested that these proteins have a role in neural cell differentiation (Calabrese et al., 2002). Stress can cause alterations in protein structure and function leading to misfolding of proteins in cells (Kaarniranta et al., 2002). One role of Hsps is to function as molecular chaperones (i.e., proteins that assist other
proteins in attaining their functional three-dimensional structures) under both normal and stressful conditions (Kaarniranta et al., 2002). Under stress induced by heat shock, Hsp chaperones promote survival by facilitating the refolding of misfolded proteins.
Heat shock protein 70 is found in the cytosol, nucleus, and endoplasmic reticulum and its expression has been detected in numerous cell populations within the nervous system, including neurons, glia and endothelial cells (Takeda et al., 2000; Calabrese et al., 2002). By embryonic (E) 15.5 in mice, Hsp70 is detectable in the central nervous system and at E17.5 all Hsps are expressed in the hippocampus (Loones et al., 2000; Calabrese et al., 2002). Developmental analysis in the postnatal rat brain has shown that basal levels increased in the cerebral hemisphere until postnatal (P) 20 and then decreased in the adult, whereas there was little change observed in the cerebellum during postnatal development (Calabrese et al., 2002). Heat shock protein 60 (Hsp60) has also been described in neurons (Calabrese et al., 2002). Developmental profiles have shown that Hsp60 increases during postnatal
development in the rat brain stem and the cerebral hemispheres; therefore Hsp60 levels are significantly higher at P20 and in adult compared to P1 in these regions (Calabrese et al., 2002).
Thioredoxin (TRX) is small protein (12-13kDa) known to function as an antioxidant protein (Holmgren, 1985). TRX has the conserved amino acid sequence Cys-Gly-Pro-Cys in its active site (Holmgren, 1985; Carvalho et al., 2006) where the two redox-active cysteines participate in reversible oxidation-reduction reactions (Holmgren, 1985). TRX appears to play biologically important roles in hormone secretion, cell signaling, regulation of the intracellular apoptotic pathway, and cell proliferation (Saitoh et al., 1998; Kobayashi-Miura et al., 2002; Tanito et al., 2002). TRX has also been shown to regulate the DNA binding
activity of various transcriptional factors such as nuclear factor (NF-kB), activator protein 1 (AP-1), myb, redox factor 1, and mitogen-activated kinase (Okamoto et al., 1992; Hirota et al., 1997). Meyer et al have reported that TRX induces AP-1 through de novo transcription of c-Fos and c-Jun, therefore leading to a possible neuroprotective function (Takagi et al., 1999). A study by Saitoh et al (1998) has shown that TRX is a physiological inhibitor of the apoptosis signal-regulating kinase (ASK) 1. TRX binds to the N-terminal region of ASK1 and inhibits the kinase activity and ASK1 dependent apoptosis (Saitoh et al., 1998). While many of the functions attributed to TRX are known to be important developmental processes, we are not aware of any specific studies of the role of TRX during neural or retinal
development.
Copper-Zinc superoxide dismutase (Cu-Zn SOD1) is an antioxidant enzyme found in the nucleus, mitochondria, and cytosol. It is abundantly expressed in all vertebrate tissue and catalyzes the conversion of superoxide anion to hydrogen peroxide (Jaarsma et al., 2000; Selverstone Valentine et al., 2005). SOD1 is considered a key enzyme to helping a cell protect itself against oxygen free radicals (Fridovich, 1995; Jaarsma et al., 2000). A mutation in the SOD1 gene causes amyotrophic lateral sclerosis (ALS), a neurodegenerative disease. To our knowledge, however, there are no published reports suggesting a potential role for SOD1 during neural or retinal development.
It is not yet clear what developmental role these stress-response proteins may have in retinal progenitor cells. However, expression of these proteins does clearly differentiate RPCs from BPCs which were generated and maintained under the same conditions and may well represent fundamental biological differences between these two cell populations.
Shared Transcriptional Regulatory Motifs in Stress-Response Genes Expressed in RPCs
Co-regulation of genes by sets of shared transcription factors is a common theme in the developmental regulation of eukaryotic gene expression (Nelander et al., 2005). Analysis of the promoter regions of the genes encoding the four stress response proteins that were over-expressed in RPCs relative to BPCs revealed 11 transcription factor-binding site motifs in common. At least 10 copies of the motifs for three of these (c-Rel, Evi-1 and Elk-1) were found within a 1 kb promoter region of each gene. Thus, transcriptional co-regulation of the genes for these four stress-related proteins is potentially responsible for their elevated expression in RPCs relative to BPCs.
C-Rel transcription factor is a member of the nuclear factor KB (NF-KB) transcription factor family. NF-KB transcription factors are composed of dimeric combinations of
members of the Rel transcription factor family (Janssens and Tschopp, 2006). Five different Rel family members have been identified in mammals: RelA, RelB, c-Rel, NF-KB1 (p50 and p105) and NF-KB2 (p52 and p100). The NF-KB/Rel proteins share a 300 amino acid long highly conserved N-terminal called the Rel homology domain (RHD) which is responsible for DNA binding and dimerization (Ravi and Bedi, 2004; Janssens and Tschopp, 2006). These transcription factors play an important role in cell proliferation, apoptosis, stress response, and inflammation.
Evi-1 is a transcription factor that encodes a 145-kDa nuclear protein of the zinc- finger family, which contains two domains of zinc fingers (Morishita et al., 1988; Matsugi et al., 1995). This transcription factor possesses seven and three repeats of Cys2His2-type zinc- finger motifs separated into two domains (Morishita et al., 1988; Hirai et al., 2001). Evi-1 also contains proline rich and acidic domains, which may function as transcriptional
activation, or repression domains (Matsugi et al., 1995). Evi-1 is thought to promote growth, cell proliferation or block differentiation in some cell types (Hirai et al., 2001).
Elk-1 is an ETS-domain transcription factor involved in the MAP (mitogen-activated protein) kinase pathway (Yang et al., 2003; Yang and Sharrocks, 2006). Phosphorylation of Elk-1 by MAP kinases triggers its activation (Yang et al., 2003). Once activated Elk-1 is converted from a transcriptional silent state to a highly active state and then back to a basal level (Yang and Sharrocks, 2006). Ternary complex factors (TCF) also represent a subclass of ETS-domain transcription factors for which Elk-1 is a member (Yang et al., 2003; Cesari et al., 2004; Yang and Sharrocks, 2006). Three TCFs have been identified in mammals, Elk- 1, SAP-1 (SRF accessory protein 1; also called Elk-4), and SAP-2 (SRF accessory protein 2; also called Elk-3) (Yang et al., 2003; Cesari et al., 2004). Cesari et al demonstrated that Elk-1 did not fulfill an essential function in mouse development, though in rats Elk-1 was
expressed selectively in neurons.
Co-regulation of preferentially RPC-expressed cellular stress-response proteins by c- Rel, Evi-1 or Elk-1 would be consistent with a role during proliferation of RPCs (a neuronal cell population). Future studies to assay the expression of these transcription factors in RPCs compared to BPCs may address this question. Certainly identification of transcription factors that underlie the differential expression of proteins between populations of cells is an
important aspect of understanding fundamental biological differences between them. Proteomics is becoming a powerful approach to identify differences in protein expression and post-translational modification between cell types (Guo et al., 2007; Ahn et al., 2007). Profiling the expression of proteins, rather than relying on analysis of differential RNA expression, is critical for understanding the many post-transcriptional mechanisms that
regulate the ultimate function of proteins in cellular processes because changes in cellular mRNA levels often do not directly correlate with changes in their protein levels (Ahn et al., 2007). Furthermore, protein expression profiling allows detection of many protein post- translational modifications and can provide valuable snapshots of cellular metabolism that complement the results of RNA-based assays.
In conclusion, we have provided the first systematic proteomics comparison of expression patterns in retinal and brain progenitor cells from genetically similar mice. The identification of four stress-response proteins among those differentially expressed in RPCs relative to BPCs is intriguing. Perhaps proliferation or differentiation of RPCs is inherently more stressful for RPCs than for BPCs, or perhaps RPCs normally have a higher baseline level of these proteins. Although there are no direct data to support either of these
possibilities at present, two of the four stress-response proteins have beenimplicated in neural development by other studies, leading us to propose that they are somehow involved in RPC development. Finally, the shared potential transcriptional regulatory sites in the genes for these four stress response proteins, suggesting their transcriptional co-regulated in RPCs, makesthese cells potentially useful as biomarkers to differentiate ‘retinal-like’ progenitor cells from ‘brain-like’ progenitor cells. Further investigation of differences between multiple populations of RPCs and BPCs during their maintenance and differentiation will further identify fundamental differences that define ‘retinal-like’ characteristics, and provide tools to assay the success of efforts to bias many populations of stem cells to adopt a retinal cell fate.
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TABLES
Identified Protein Spots Over-expressed in Retinal Progenitor Cells When Compared to Brain Progenitor Cells
Accession Number Protein Identifications Expression Fold Increased in RPCs compared to BPCs
P63017 Heat shock cognate 71 kDa protein
Unique
P14733 Lamin B1 + 2.408
P63038 60 kDa heat shock protein, mitochondrial precursor
(Hsp60)
+ 2.015 P61979 Heterogeneous nuclear
ribonucleoprotein K Unique P47753 F-actin capping protein alpha-
1 subunit
Unique Q99PF4 Cadherin-23 precursor
(Otocadherin)
Unique
P57776 Elongation factor 1-delta Unique P67778 Prohibitin (B-cell receptor
associated protein 32) (BAP 32)
+ 2.093
P10639 Thioredoxin + 2.802
P16045 Galectin-1 (Beta-galactoside- binding lectin L-14-I)
+ 2.973 P08228 Superoxide dismutase [Cu-
Zn] (EC 1.15.1.1) + 3.313 P62962 Profilin-1 + 2.459 P49312 Heterogeneous nuclear ribonucleoprotein A1 + 2.451 P08249 Malate dehydrogenase, mitochondrial precursor + 2.711 O35737 Heterogeneous nuclear
ribonucleoprotein H (hnRNP H)
+ 2.108 O70251 Elongation factor 1-beta (EF-
1-beta) + 2.371
P29758 Ornithine aminotransferase, mitochondrial precursor (EC
2.6.1.13)
Unique P56959 RNA-binding protein FUS
(Pigpen protein) + 2.287 P60710 Actin, cytoplasmic 1 (Beta-
actin) Unique
P31786 Acyl-CoA-binding protein (ACBP) (Diazepam binding
inhibitor) (DBI) (Endozepine)
Unique Q9D051 Pyruvate dehydrogenase E1
component beta subunit, mitochondrial precursor
Unique Q9DBJ1 Phosphoglycerate mutase 1
(EC 5.4.2.1)
+ 5.498
Table 1. List of protein identifications and expression levels between retinal progenitor cells (RPCs) and brain progenitor cells (BPCs).
Protein GO: Biological Process GO: Molecular
Function GO: Cellular Localization
Phosphoglycerate mutase 1 1.glycolysis 2.metabolism 1.bisphosphoglycerate mutase activity 2.catalytic activity 3.hydrolase activity 4.intramolecular transerase activity, phosphotransferases 5.isomerase activity 6. phosphoglycerate mutase activity 1.cytosol
RNA-binding protein FUS (Pigpen protein)
1.positive regulation of transcription from RNA polymerase II promoter
1.transcriptional activator activity
2.DNA binding 3.nucleic acid binding 4.RNA binding 5.zinc ion binding
1.cytoplasm 2.nucleus
60 kDa heat shock protein,
mitochondrial precursor (Hsp60) 1.cellular protein metabolism 2.protein folding 1.ATP binding 2.protein binding 3.unfolded protein binding
1.mitochondrion Malate dehydrogenase,
mitochondrial precursor 1.glycolysis 2.malate metabolism 3.tricarboxylic acid cycle intermediate metabolism 1.L-malate dehydrogenase activity 2.malate dehydrogenase activity 3.oxidoreductase activity 1.mitochondrion Heterogeneous nuclear ribonucleoprotein A1 (Helix- destabilizing protein) 1.mRNA processing 2.mRNA-nucleus export 3.RNA processing 4.transport
1.nucleic acid binding
2.RNA binding 1.nucleus 2.ribonucleoprotein complex
Acyl-CoA-binding protein (ACBP) (Diazepam binding inhibitor) (DBI) (Endozepine)
1.transport 1.acyl-CoA binding
2.lipid binding
1.mitochondrion
Lamin B1 1.structural molecule activity 1.lamin filament
2.nucleus
3.intermediate filament Prohibitin (B-cell receptor
associated protein 32) (BAP32)
1.DNA metabolism 1.mitochondrion
Thioredoxin 1.electron transport
2.transport
1.electron transporter activity 1.mitochondrion Superoxide dismutase 1. activation of MAPK
2.DNA fragmentation during apoptosis
3.response to oxidative stress 4.removal of superoxide radicals
5.superoxide metabolism
1.copper, zinc superoxide dismutase activity 2.antioxidant activity 3.metal ion binding 4.oxidoreductase activity 5.superoxide dismutase activity
Protein GO: Biological Process GO: Molecular
Function GO: Cellular Localization
Profilin-1 1.neural tube closure
2.regulation of transcription from RNA polymerase II promoter 3.regulation of actin polymerization and/or depolymerization 4.sequestering of actin monomers 5.actin cytoskeleton organization and biogenesis 6.cytoskeleton organization and biogenesis 1.protein binding 2.actin binding 1.cytoplasm 2.nucleus 3.actin cytoskeleton 4.cytoskeleton Heterogeneous nuclear ribonucleoprotein H (hnRNP H)
1.nucleic acid binding
2.RNA binding
1.nucleus
2.ribonucleoprotein complex Heat shock cognate 71 kDa
protein (Heat shock 70 kDa protein 8)
1.chaperone cofactor dependent protein folding 2.protein folding 3.regulation of cell cycle 4.response to unfolded protein
1.ATPase activity, coupled