Quantitative modulation of thyroid iodide peroxidase by thyroid stimulating hormone

The activity of rat thyroid iodide peroxidase fell to 8% of the normal value 48 hours after hypophysectomy. Rats given injections of thyroid stimulating hormone manifested an enzyme activity indistinguishable from that of the sham-operated animals. Cycloheximide prevented the thyroid stimulating hormone-induced restoration of the enzyme activity. The incorporation of ¹⁴C-leucine into the thyroid gland decreased gradually and reached two thirds of the sham-operated group by 48 hours after hypophysectomy. Thyroid stimulating hormone administration prevented this decrease, as observed for iodide peroxidase activity. Thyroidal RNA contents decreased also in hypophysectomized rats, thyroid stimulating hormone treatment prevented the reduction of RNA contents and no significant change was observed in thyroidal DNA contents. These data are consistent with the idea that protein biosynthesis is involved in thyroid stimulating hormone regulation of thyroidal iodide peroxidase and that the life span of the peroxidase is less than 48 hours.     

Quantitative proteomics combined with BAC TransgeneOmics reveals in vivo protein interactions

Protein interactions are involved in all cellular processes. Their efficient and reliable characterization is therefore essential for understanding biological mechanisms. In this study, we show that combining bacterial artificial chromosome (BAC) TransgeneOmics with quantitative interaction proteomics, which we call quantitative BAC–green fluorescent protein interactomics (QUBIC), allows specific and highly sensitive detection of interactions using rapid, generic, and quantitative procedures with minimal material. We applied this approach to identify known and novel components of well-studied complexes such as the anaphase-promoting complex. Furthermore, we demonstrate second generation interaction proteomics by incorporating directed mutational transgene modification and drug perturbation into QUBIC. These methods identified domain/isoform-specific interactors of pericentrin- and phosphorylation-specific interactors of TACC3, which are necessary for its recruitment to mitotic spindles. The scalability, simplicity, cost effectiveness, and sensitivity of this method provide a basis for its general use in small-scale experiments and in mapping the human protein interactome.     

Determination of nuclear receptor corepressor interactions with the thyroid hormone receptor

The thyroid hormone receptor (TR) recruits the nuclear corepressors, nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid hormone receptors (SMRT), to target DNA elements in the absence of ligand. While the TR preferentially recruits NCoR, the mechanism remains unclear. The corepressors interact with the TR via interacting domains (IDs) present in their C terminus which contain a conserved motif termed a CoRNR box. Despite their similarity, the corepressor IDs allow for nuclear receptor specificity. Here we demonstrate that NCoR stabilizes the TR homodimer when bound to DNA by preventing its dissociation from thyroid hormone response elements. This suggests that NCoR acts to hold the repression complex in place on target elements. The TR homodimer recruits NCoR through two of its three IDs, one of which is not present in SMRT. This unique ID, N3, contains a CoRNR box but lacks the extended helical motif present in each of the other IDs. Instead, N3 contains an isoleucine just proximal to this motif. This isoleucine is also conserved in N2 but not in the corresponding S2 domain in SMRT. On thyroid hormone response elements and in mammalian cells this residue is critical in both N3 and N2 for high-affinity TR binding. In addition, this residue also controls specificity for the interactions of TR with NCoR. Together these data suggest that the specific recruitment of NCoR by the TR through a unique motif allows for stabilization of the repression complex on target elements.     

Quantitative plant proteomics

Quantitation is an inherent requirement in comparative proteomics and there is no exception to this for plant proteomics. Quantitative proteomics has high demands on the experimental workflow, requiring a thorough design and often a complex multi-step structure. It has to include sufficient numbers of biological and technical replicates and methods that are able to facilitate a quantitative signal read-out. Quantitative plant proteomics in particular poses many additional challenges but because of the nature of plants it also offers some potential advantages. In general, analysis of plants has been less prominent in proteomics. Low protein concentration, difficulties in protein extraction, genome multiploidy, high Rubisco abundance in green tissue, and an absence of well-annotated and completed genome sequences are some of the main challenges in plant proteomics. However, the latter is now changing with several genomes emerging for model plants and crops such as potato, tomato, soybean, rice, maize and barley. This review discusses the current status in quantitative plant proteomics (MS-based and non-MS-based) and its challenges and potentials. Both relative and absolute quantitation methods in plant proteomics from DIGE to MS-based analysis after isotope labeling and label-free quantitation are described and illustrated by published studies. In particular, we describe plant-specific quantitative methods such as metabolic labeling methods that can take full advantage of plant metabolism and culture practices, and discuss other potential advantages and challenges that may arise from the unique properties of plants.     

Quantitative proteomics of lymphocytes

Lymphocytes are the best-studied higher eukaryote cells. In this report, quantitative relationships of the protein components in resting cell, blast cell and plasma cell types are evaluated. The comparison of these cell types leads to the conclusion that resting cells synthesize about one-twentieth of the protein species as compared to blast cells. Blast cells seem to be metabolically the most robust lymphocyte type. Plasma cells are geared towards synthesis of one main product (antibody in B plasma cells), while most of the synthesis of other protein species (including those for housekeeping and repair) decreases as the messages decay. Although the data presented in this communication allow a meaningful comparison of three cell populations, they are far from providing a full picture. Both silver staining and radiofluorography depict only proteins of high or intermediate abundance. Silver staining misses most proteins present at <10 000 copies/cell, while radiofluorography misses all those proteins with slow turnover (and those with no methionine residue in their sequence). The detection of 1100 spots in the blast cell-related radiofluorograph includes visualization of some 97-99% of protein mass, but some 3900 polypeptide species in the remaining 1-3% of protein mass will pass undetected. This protein mass (0.7-2 pg) reflects some 2500-7500 copies of each of those 3900 polypeptide species that are present in the cell below the detection limit. The work emphasizes that full understanding of cellular function can be achieved only if quantitative aspects of cell inventory are considered.     

Quantitative proteomics of complex mixtures

Measurement of biologically important effector protein molecules has been a long-standing essential component of biological research. Advances in biotechnology, in the form of high-resolution mass spectrometers, and in bioinformatics, now allow the simultaneous quantitative analysis of thousands of proteins. While these techniques still do not allow definitive identification of the entire proteome of complex mixtures, such as cells, quantitative analyses of hundreds to thousands of proteins in such complex mixtures provides a means to elucidate molecular alterations that occur during perturbation of cellular systems. This article will outline considerations of reducing sample complexity, by strategies such as multidimensional separations (gel-based and chromatography-based, including multidimensional protein identification technology). In addition, some of the most common methods used to quantitatively measure proteins in complex mixtures (2D difference in-gel electrophoresis, isotope-coded affinity tags, isotope-coded protein labeling, tandem mass tags, isobaric tags for relative and absolute quantitation, stable isotope labeling of amino acids in cell culture and label-free), as well as recent examples of each strategy, are described.     

Molecular modeling of the thyroid hormone interactions with alpha v beta 3 integrin

A cell surface receptor for thyroid hormone has recently been identified on the extracellular domain of integrin alphavbeta3. In a variety of human and animal cell lines this hormone receptor mediates activation by thyroid hormone of the cellular mitogen-activated protein kinase (MAPK) signal transduction cascade. An arginine-glycine-aspartate (RGD) recognition site on the heterodimeric integrin is essential to the binding of a variety of extracellular matrix proteins. Recent competition data reveal that RGD peptides block hormone-binding by the integrin and consequent MAPK activation, suggesting that the hormone interaction site is located at or near the RGD recognition site on integrin alphavbeta3. A deaminated thyroid hormone (l-thyroxine, T4) analogue, tetraiodothyroacetic acid (tetrac, T4ac), inhibits binding of T4 and 3,5,3'-triiodo-l-thyronine (T3) to alphavbeta3, but does not activate MAPK. Structural data show that the RGD cyclic peptide binds at the interface of the propeller of the alphav and the B domains on the integrin head [Xiong JP, Stehle T, Zhang R, Joachimiack A, Frech M, Goodman SL, et al. Crystal structure of the extracellular segment of integrin alphavbeta3 in complexing with an Arg-Gly-Asp ligand. Science 2002;296:151-5]. To model potential interactions of thyroid hormone analogues with integrin, we mapped T4 and T4ac to the binding site of the RGD peptide. Modeling studies indicate that there is sufficient space in the cavity for the thyroid hormone to bind. Since the hormone is smaller in overall length than the RGD peptide, the hormone does not interact with the Arg recognition site in the propeller domain from alphav. In this model, most of the hormone interactions are with betaA domain of the integrin. Mutagenic studies can be carried out to validate the role of these residues in directing hormone interactions.     

Quantitative proteomics analysis of the secretory pathway

We report more than 1400 proteins of the secretory-pathway proteome and provide spatial information on the relative presence of each protein in the rough and smooth ER Golgi cisternae and Golgi-derived COPI vesicles. The data support a role for COPI vesicles in recycling and cisternal maturation, showing that Golgi-resident proteins are present at a higher concentration than secretory cargo. Of the 1400 proteins, 345 were identified as previously uncharacterized. Of these, 230 had their subcellular location deduced by proteomics. This study provides a comprehensive catalog of the ER and Golgi proteomes with insight into their identity and function.     

Quantitative proteomics using mass spectrometry

The use of stable isotopes as internal standards in mass spectrometry has opened a new era for quantitative proteomics. Depending on the point at which the label is introduced, most procedures can be classified as in vivo labeling, in vitro pre-digestion labeling or in vitro post-digestion labeling. In vivo labeling has been used for cells that can be grown in culture and has the advantage of being more accurate. The pre-digestion and post-digestion labeling procedures are suitable for all types of sample including human body fluids and biopsies. Several new mass spectrometric strategies mark significant achievements in determining relative protein concentrations and in quantifying post-translational modifications. However, further technology developments are needed for understanding the complexity of a dynamic system like the proteome.     

Quantitative proteomics of intracellular Porphyromonas gingivalis

Whole-cell quantitative proteomic analyses were conducted to investigate the change from an extracellular to intracellular lifestyle for Porphyromonas gingivalis, a Gram-negative intracellular pathogen associated with periodontal disease. Global protein abundance data for P. gingivalis strain ATCC 33277 internalized for 18 h within human gingival epithelial cells and controls exposed to gingival cell culture medium were obtained at sufficient coverage to provide strong evidence that these changes are profound. A total of 385 proteins were overexpressed in internalized P. gingivalis relative to controls; 240 proteins were shown to be underexpressed. This represented in total about 28% of the protein encoding ORFs annotated for this organism, and slightly less than half of the proteins that were observed experimentally. Production of several proteases, including the classical virulence factors RgpA, RgpB, and Kgp, was decreased. A separate validation study was carried out in which a 16-fold dilution of the P. gingivalis proteome was compared to the undiluted sample in order to assess the quantitative false negative rate (all ratios truly alternative). Truly null (no change) abundance ratios from technical replicates were used to assess the rate of quantitative false positives over the entire proteome. A global comparison between the direction of abundance change observed and previously published bioinformatic gene pair predictions for P. gingivalis will assist with future studies of P. gingivalis gene regulation and operon prediction.     

Thyroid hormone-divalent cation interactions. Effect of thyroid hormone on mitochondrial calcium metabolism

The effect of l-triiodothyronine (T₃) on calcium metabolism was studied in mitochondria isolated from thyroidectomized rats. The maximal rate of low-level calcium uptake (V) was enhanced by T₃ in the absence or the presence of added acetate; the $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ value was unchanged. Further, the rate constant (K_e) derived from measurement of the efflux of calcium was stimulated 10-fold upon the addition of T₃. This stimulating effect was not seen in the presence of acetate. In studies of adenine nucleotide translocation, calcium stimulated the translocation of ATP out of these mitochondria, but only in the presence of T₃. These results suggest that T₃ alters calcium ion mobility within the mitochondrial membrane and hence modulates calcium-dependent metabolic processes. The precise locus of this affect remains to be identified.     

Quantitative proteomics to decipher ubiquitin signaling

Ubiquitin signaling plays an essential role in controlling cellular processes in eukaryotes, and the impairment of ubiquitin regulation contributes to the pathogenesis of a wide range of human diseases. During the last decade, mass spectrometry-based proteomics has emerged as an indispensable approach for identifying the ubiquitinated proteome (ubiquitinome), ubiquitin modification sites, the linkages of complex ubiquitin chains, as well as the interactome of ubiquitin enzymes. In particular, implementation of quantitative strategies allows the detection of dynamic changes in the ubiquitinome, enhancing the ability to differentiate between function-relevant protein targets and false positives arising from biological and experimental variations. The profiling of total cell lysate and the ubiquitinated proteome in the same sets of samples has become a powerful tool, revealing a subset of substrates that are modulated by specific physiological and pathological conditions, such as gene mutations in ubiquitin signaling. This strategy is equally useful for dissecting the pathways of ubiquitin-like proteins.     

Dominant inhibition of thyroid hormone action selectively in the pituitary of thyroid hormone receptor-beta null mice abolishes the regulation of thyrotropin by thyroid hormone

Thyroid hormones, T4 and T3, regulate their own production by feedback inhibition of TSH and TRH synthesis in the pituitary and hypothalamus when T3 binds to thyroid hormone receptors (TRs) that interact with the promoters of the genes for the TSH subunit and TRH. All TR isoforms are believed to be involved in the regulation of this endocrine axis, as evidenced by the massive dysregulation of TSH production in mice lacking all TR isoforms. However, the relative contributions of TR isoforms in the pituitary vs. the hypothalamus remain to be completely elucidated. Thus, to determine the relative contribution of pituitary expression of TR-alpha in the regulation of the hypothalamic-pituitary-thyroid axis, we selectively impaired TR-alpha function in TR-beta null mice (TR-beta-/-) by pituitary restricted expression of a dominant negative TR-beta transgene harboring a delta337T mutation. These animals exhibited 10-fold and 32-fold increase in T4 and TSH concentrations, respectively. Moreover, the negative regulation of TSH by exogenous T3 was completely absent and a paradoxical increase in TSH concentrations and TSH-beta mRNA was observed. In contrast, prepro-TRH expression levels in T3-treated TR-beta-/- were similar to levels observed in the delta337/TR-beta-/- mice, and ligand-independent activation of TSH in hypothyroid mice was equivalently impaired. Thus, isolated TR-beta deficiency in TRH paraventricular hypothalamic nucleus neurons and impaired function of all TRs in the pituitary recapitulate the baseline hormonal disturbances that characterize mice with complete absence of all TRs.