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Results Here we identify components of the proteome and phosphoproteome in the tail fin that changed within 48 h of exposure of premetamorphic Rana catesbeiana tadpoles to 10 nM 3,5,3'-triiodothyronine (T 3). To this end, we developed a cell and protein fractionation method combined with two-dimensional gel electrophoresis and phosphoprotein-specific staining. Altered proteins were identified using mass spectrometry (MS). We identified and cloned a novel Rana larval type I keratin, RLK I, which may be a target for caspase-mediated proteolysis upon exposure to T 3. In addition, the RLK I transcript is reduced during T 3-induced and natural metamorphosis which is consistent with a larval keratin. Furthermore, GILT, a protein involved in the immune system, is changed in phosphorylation state which is linked to its activation. Using a complementary MS technique for the analysis of differentially-expressed proteins, isobaric tags for relative and absolute quantitation (iTRAQ) revealed 15 additional proteins whose levels were altered upon T 3 treatment.
The success of identifying proteins whose levels changed upon T 3 treatment with iTRAQ was enhanced through de novo sequencing of MS data and homology database searching. These proteins are involved in apoptosis, extracellular matrix structure, immune system, metabolism, mechanical function, and oxygen transport. Conclusion We have demonstrated the ability to derive proteomics-based information from a model species for postembryonic development for which no genome information is currently available. The present study identifies proteins whose levels and/or phosphorylation states are altered within 48 h of the induction of tadpole tail regression prior to overt remodeling of the tail. In particular, we have identified a novel keratin that is a target for T 3-mediated changes in the tail that can serve as an indicator of early response to this hormone.
Thyroid hormones (TH) are important signaling molecules in vertebrates that regulate homeostasis, growth, and development. One developmental process that is dependent upon the presence of TH is amphibian metamorphosis. During metamorphosis the larval, aquatic, herbivorous tadpole transforms into a terrestrial, carnivorous juvenile frog. This event requires drastic changes in essentially every organ and tissue of the tadpole and includes: the resorption of larval organs and tissues, remodeling of larval organs into juvenile form, and de novo development of organs and tissues. Metamorphosis is completely controlled through the control of serum TH levels. The thyroid gland mainly produces the thyroid hormone, 3,5,3',5'-tetraiodothyronine (T 4 or thyroxine), which is converted into the biologically more active form, 3,5,3'-triiodothyronine (T 3), in the peripheral tissues ,.
Progression through natural metamorphosis is dependent upon a tightly-controlled rise and fall in TH levels. Premetamorphic tadpoles are functionally athyroid with no measurable levels of THs. TH levels gradually increase during prometamorphosis and reach maximal levels at metamorphic climax. At this stage, overt remodelling of the tadpole rapidly ensues. Premetamorphic tadpoles can be induced to undergo precocious metamorphosis by exposure to TH. The best understood mechanism of TH action involves TH binding to nuclear thyroid hormone receptors (TRs) which regulate gene expression ,. TH binding to TRs can either activate or repress transcription of responsive genes through recruitment of coactivators and corepressors, respectively ,.
Differential nuclear TR levels and intracellular T 3 levels controlled by deiodinase activity and TH-binding proteins, contribute to tissue-specific responses ,. However, the response to TH also depends on the existing complement of other proteins that can influence cell fate (e.g.
Regression of the tail versus growth and differentiation of the hindlimb). However, the molecular mechanisms are poorly understood. Most TH-responsive genes are up-regulated and these have been most commonly studied particularly in the tail –. The genetic program required for tail regression is established between 24 and 48 h of TH exposure at a 'commitment point' after which removal of TH or exposure to transcription or protein synthesis inhibitors cannot prevent regression ,. Studies based on PCR subtractive hybridization methods and cDNA gene arrays have identified a number of possible genes involved in this process –.
However, the relationship of most of these genomic findings to changes in the proteome has yet to be identified and there is growing evidence for non-classical TH action through phosphorylation signaling pathways –. Proteomic scale studies on TH-dependent tail regression have been scarce. Identified several 35S-methionine labelled proteins from Rana catesbeiana tail fin that change during natural and precocious metamorphosis using two-dimensional (2D) gel analysis. Kobayashi et al.
Used 2D gel electrophoresis to analyze changes in protein expression in the back and tail skin of Xenopus laevis during metamorphosis. From the 2D protein spot patterns they could classify the back skin into larval or adult type and observe the transition.
Attempts were made in these studies to identify the altered protein spots. This, however, involved identifying the spots based on position, comigration or immunological detection methods. Using 2D gel separation and mass spectrometry (MS) for protein identification, we were able to identify 9 proteins whose expression was altered in the X.
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Laevis tadpole tail during TH-induced metamorphosis. Regardless of the approach, the lack of any sample fractionation led to the identification of only abundant proteins. In this work we identified novel changes in the proteome and phosphoproteome associated with the induction of T 3-dependent tail regression of Rana catesbeiana tadpoles. Proteins that changed in abundance were detected using two-dimensional (2D) gel electrophoresis and isobaric tags for relative and absolute quantitation (iTRAQ) methods. Alterations in phosphorylation were revealed using a phosphoprotein-specific stain. Mass spectrometry (MS) analyses were then used for protein identification. Proteome coverage was enhanced through the use of cell and protein fractionation to reveal several proteins that are altered within 48 h of T 3 exposure.
We developed two separate procedures based on differential centrifugation to generate nuclear and cytosolic/mitochondrial/microsomal fractions (Fig. The nuclear extraction procedure was optimized to minimize nuclear clumping, increase nuclei stability, and minimize cytoskeletal, cytoplasmic/organelle and DNA contamination.
The cytosolic/mitochondrial/microsomal extraction procedure was developed to increase the disruption of tail fin cell membrane and increase mitochondrial stability and purity. The mitochondria-enriched fraction was obtained with a 12,000 × g centrifugation and it likely also contained lysosomes, peroxisomes, Golgi and endoplasmic reticulum (ER). Centrifugation at 100,000 × g removed the vesicles of the plasma membrane, endosomes, Golgi and ER into the microsomal pellet leaving the cytosolic supernatant with its soluble molecules (cytosolic fraction; Fig.
Nuclear integrity was monitored by microscopy (data not shown) and fractionation efficiency was determined using immunoblot analysis for subcellular markers. An immunoblot for cytochrome c (a mitochondrial marker) shows substantial enrichment for that organelle in the mitochondrial fraction (Fig. ) while an immunoblot for the nuclear markers, lamins B1 and B2, shows enrichment of nuclei in the nuclear fraction (Fig.
Figure 1 Subcellular fractionation of the tail fin proteome. (A) Fractionation of tail fin cells into subcellular compartments and subsequent treatments of those fractions. Two different extraction procedures, based on differential centrifugation, were developed to generate the nuclear and the cytoplasmic/mitochondrial/microsomal fractions. (B) SDS-PAGE shows the successful fractionation of the total tail fin proteome into the cytosolic (Cytos), mitochondrial (Mito), microsomal (Micros), and nuclear (Nucl) fractions. Relative molecular weights of protein standards are indicated in kDa.
(C) Immunoblot of the gel in (B) for the mitochondrial marker, cytochrome c (arrow) showing the enrichment of mitochondria in the expected fraction. (D) Immunoblot of the gel in (B) for the nuclear markers, lamin B1 and B2 (double arrow) showing the enrichment of nuclei in the expected fraction. The cytosolic fraction is a complex mixture of many proteins and was therefore further fractionated using anion-exchange high performance liquid chromatography (HPLC) (Fig. A step-gradient anion-exchange HPLC procedure was developed that used ammonium bicarbonate as a volatile buffer in place of commonly used salt and non-volatile buffer to provide salt-free fractions after lyophilization to render the samples compatible with the subsequent 2D gel analysis.
The cytosolic fraction was thus further fractionated into five fractions: 40 mM (unbound proteins), 190 mM, 260 mM, 340 mM and 1 M ammonium bicarbonate with each fraction (except 40 mM) yielding roughly equal amounts of protein as shown by SDS-PAGE (Fig. Proteins within each of the resulting fractions were then separated by 2D polyacrylamide gel electrophoresis which separates proteins based on their molecular weight and isoelectric point (pI). Therefore, the entire fractionation protocol divided the tail fin proteome over eight 2D gels: nuclear, mitochondrial, microsomal (Fig. ) and five cytosolic fractions (Fig.
This fractionation method increases the ability to observe expression changes in low abundance proteins and provides information on subcellular localization of proteins which cannot be achieved by examining a whole cell homogenate on a single 2D gel. From our results, it is evident that each of the fractions shows a distinctive pattern of spots with many unique spots per fraction analyzed (Figs.
There is some overlap with the more abundant protein spots between the neighboring fractions of the HPLC separation and between the microsomal and mitochondrial fractions, which probably share many cellular membrane compartments (e.g. Golgi, ER, and lysosomes). Phosphoproteins were detected on the 2D gels with a phosphoprotein-specific fluorescent stain (Pro-Q Diamond) which detects phosphorylation on Ser, Thr and Tyr residues (Figs.
Total proteins and phosphoproteins were detected in the same 2D gel allowing for easy identification of phosphoprotein location and subsequent isolation for MS analysis. Figure 2 Anion-exchange HPLC fractionation of the cytosolic fraction. (A) The cytosolic fraction was further fractionated using an anion-exchange column (Accell QMA) with a step-gradient of increasing concentrations of ammonium bicarbonate (straight lines). The concentrations are indicated on each step while absorbance was measured at 280 nm indicating the protein yield of each fraction. (B) The Coomassie blue-stained SDS-PAGE gel shows the fractionation of the cytosolic sample (total) with the lanes corresponding to the cytosolic fractions below the peaks of the HPLC chromatogram.
Note the resulting enrichment of certain protein bands. Relative molecular weights of protein standards are indicated in kDa. Figure 4 2D gel analysis of the anion-exchange HPLC cytosolic fractions. Proteins from each of the fractions resulting from the anion-exchange HPLC of the cytosolic sample were separated by 2D-PAGE according to molecular weight and pI point. The 40 mM fraction is the unbound protein fraction, while the subsequent fractions are proteins eluted by the increasing ammonium bicarbonate concentration step-gradients. Total proteins were detected by colloidal Coomassie stain while phosphoproteins were detected in the same gel using the ProQ Diamond phosphoprotein-specific stain. Relative molecular weights of protein standards are indicated in kDa.
The above methods were used to analyze the proteome and phosphoproteome of the premetamorphic R. Catesbeiana tail fin undergoing precocious metamorphosis at 24 and 48 h induced with 10 nM T 3. A minimum of three independent replicates allowed for the verification of changes in protein and phosphoprotein expression and MS analysis was used for protein identification. Identification of a unique R. Catesbeianakeratin fragment. A prominent protein spot at 24 kDa and pI 5 was increased upon T 3 treatment on the 2D gels of several fractions (Fig.
It was observed in the 340 mM cytoplasmic fraction as well as in the microsomal, mitochondrial and nuclear fractions. This protein spot was increased by 2–3 fold as early as 24 h (data not shown), but was more intensely expressed at 48 h (2.6 to 5.1 fold increase depending on the fraction) (Fig. The greatest increase was observed in the microsomal fraction. The protein spots from each of the fractions were separately analyzed by mass spectrometry proving that each fraction represented the same protein. A combination of electrospray-ionization quadrupole time-of-flight (ESI-QqTOF) and matrix-assisted laser desorption ionization TOF-TOF (MALDI-TOF-TOF) tandem-MS (MS/MS) analyses allowed for peptide sequence information to be obtained for 11 different peptides from this protein (Table ). Protein database searches with these peptides gave the highest homology match to the X.
Laevis type I keratin 47 kDa protein NCBI: P05781 also known as X. Laevis keratin B2 NCBI: 1304283B) from the XK81 gene family. Figure 5 Identification of a novel R. Catesbeiana type I (RLK I) keratin fragment by 2D gel analysis.
(A) 2D gel regions of the 340 mM cytosolic, microsomal, mitochondrial, and nuclear fractions show the increase of a protein spot at 24 kDa and pI 5 due to T 3 treatment at 48 h. The corresponding gel region, stained with a phosphoprotein stain, is shown for the nuclear fraction revealing additional changes in the phosphoproteome. The white arrows indicate the spot identified as a novel type I keratin RLK I fragment in the T 3 samples (see Table 1). In the phosphoprotein gel, the white arrow indicates a possible phosphorylated form of the keratin fragment. The gray arrows indicate an additional unidentified protein and phosphoproteins that are altered upon T 3 treatment. Relative molecular weights of protein standards are indicated in kDa.
(B) Spot density measurements (in arbitrary values) are graphed for the corresponding 2D gels on the left. The white bar represents the control while the gray bar represents the T 3 treatment. Error bars represent the standard error of the mean from three independent controls and three independent T 3 samples. Significance is indicated by an asterisk for p.
Observed peptide mass (Da, M+H +) 1 Peptide sequence from MS/MS 2 Identified by MALDI-TOF-TOF 3% confidence 4(MS/MS/MALDI) Matched database sequence 5 807.4 LAADDFR Yes 84/89 LAADDFR 809.4 LASYLDK Yes 100/na LASYL EK 991.5 FENELALR Yes 100/98 FENELALR 1041.6 LVLQIDNAR Yes 100/100 VVLQIDNA K 1073.6 ILAATIDNSR Yes 100/100 IL SATIDNSR 1079.5 VLDELTMSR Yes 100/74 VLDELT LAR 1184.6 YYDIINDLR - 96/- Y FEII SDLR 1202.6 QSVEADINGLR - 43/- QSVE TDINGLR 1224.6 NHEEELQVAR - 73/- NHEEE MSIA K 1232.7 - Yes -/100 LKFENELALR 1301.6 ALEAANTELELK - 93/- ALEAAN ADLELK. 1Observed peptide masses resulting from the tryptic digestion of the protein spot, reported as singly charged. 2Peptide sequence information deduced from MS/MS spectra of the corresponding peptides from ESI-QqTOF analysis. The masses of isoleucine are indistinguishable from leucine in MS and therefore L can be I and vice versa. 3Indicates which peptides were additionally observed with MALDI-TOF-TOF analysis. 4Percent confidence for the peptide sequences as reported by PEAKS software for the ESI-QqTOF spectra and by MASCOT for MALDI-TOF-TOF data.
5Highest homology match from protein database searching with the observed peptide sequences to X. Laevis type I keratin 47 kD using SPIDER software. Bold lettering indicates differences between the observed and database sequences. Parts of the amino acid sequence from two peptides (ALEAANTELELK and NHEEELQVAR) flanking the majority of the peptide sequence identified were used to generate degenerate primers. Degeneracy was limited by taking into account codon usage bias for R. Catesbeiana and other identified type I keratin cDNA sequences.
Two primers with 32 fold degeneracy each generated a single 380 bp PCR product from R. Catesbeiana tail cDNA. Based on this sequence two gene specific primers (GSP) were designed to perform 5'- and 3'-rapid amplification of cDNA ends (RACE). Two overlapping clones were obtained from the 5'- and 3'-RACE containing the entire open reading frame of this keratin gene (Fig. The cloned sequence was 1728 bp long, with a 109 bp 3'-untranslated region, and a polyadenylation signal, AATAAA, at 17 nucleotides upstream of the poly(A) tract.
The deduced amino acid sequence coded for a 481 amino acid protein (predicted size of 52 kDa and pI 5.0) and matched exactly all of the observed peptides from the MS analysis indicating that the correct corresponding cDNA sequence was cloned. BLASTp analysis and ClustalW alignment with this 481 amino acid sequence revealed the highest identity and similarity (80 and 90%, respectively) with the X.
Laevis type I keratin 47 kDa protein NCBI: P05781 (Fig. About a dozen keratin proteins have been identified for X.