The tetrameric protein transthyretin dissociates to a non-native monomer in solution. A novel model for amyloidogenesis.
(17/714)
In amyloidosis, normally innocuous soluble proteins polymerize to form insoluble fibrils. Amyloid fibril formation and deposition have been associated with a wide range of diseases, including spongiform encephalopathies, Alzheimer's disease, and familial amyloid polyneuropathies (FAP). In certain forms of FAP, the amyloid fibrils are mostly constituted by variants of transthyretin (TTR), a homotetrameric plasma protein implicated in the transport of thyroxine and retinol. The most common amyloidogenic TTR variant is V30M-TTR, and L55P-TTR is the variant associated with the most aggressive form of FAP. Recently, we reported that TTR dissociates to a monomeric species at pH 7.0 and nearly physiological ionic strengths (Quintas, A., Saraiva, M. J., and Brito, R. M. (1997) FEBS Lett. 418, 297-300). Here, we show that the tetramer dissociation is apparently irreversible; and based on intrinsic tryptophan fluorescence and fluorescence quenching experiments, we show that the monomeric species formed upon tetramer dissociation is non-native. We also show, based on 1-anilino-8-naph-thalenesulfonate binding studies, that this monomeric species appears not to behave like a molten globule. These data allowed us to propose a model for TTR amyloidogenesis based on tetramer dissociation occurring naturally under commonly observed physiological solution conditions. (+info)
Long-wavelength iodide-sensitive fluorescent indicators for measurement of functional CFTR expression in cells.
(18/714)
Limitations of available indicators [such as 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ)] for measurement of intracellular Cl(-) are their relatively dim fluorescence and need for ultraviolet excitation. A series of long-wavelength polar fluorophores was screened to identify compounds with Cl(-) and/or I(-) sensitivity, bright fluorescence, low toxicity, uniform loading of cytoplasm with minimal leakage, and chemical stability in cells. The best compound found was 7-(beta-D-ribofuranosylamino)-pyrido[2, 1-h]-pteridin-11-ium-5-olate (LZQ). LZQ is brightly fluorescent with excitation and emission maxima at 400-470 and 490-560 nm, molar extinction 11,100 M(-1). cm(-1) (424 nm), and quantum yield 0.53. LZQ fluorescence is quenched by I(-) by a collisional mechanism (Stern-Volmer constant 60 M(-1)) and is not affected by other halides, nitrate, cations, or pH changes (pH 5-8). After LZQ loading into cytoplasm by hypotonic shock or overnight incubation, LZQ remained trapped in cells (leakage <3%/h). LZQ stained cytoplasm uniformly, remained chemically inert, did not bind to cytoplasmic components, and was photobleached by <1% during 1 h of continuous illumination. Cytoplasmic LZQ fluorescence was quenched selectively by I(-) (50% quenching at 38 mM I(-)). LZQ was used to measure forskolin-stimulated I(-)/Cl(-) and I(-)/NO(-)(3) exchange in cystic fibrosis transmembrane conductance regulator (CFTR)-expressing cell lines by fluorescence microscopy and microplate reader instrumentation using 96-well plates. The substantially improved optical and cellular properties of LZQ over existing indicators should permit the quantitative analysis of CFTR function in gene delivery trials and high-throughput screening of compounds for correction of the cystic fibrosis phenotype. (+info)
Expression and intracellular processing of chimeric and mutant CFTR molecules.
(19/714)
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-activated chloride channel comprising two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs) and a unique regulatory (R) domain. The most frequent cystic fibrosis (CF) mutation, a deletion of Phe508 in NBD1, results in the retention of the DeltaF508 CFTR in the endoplasmic reticulum, as do many other natural or constructed mutations located within the first NBD. In order to further define the role of NBD1 in CFTR folding and to determine whether the higher frequency of mutations in NBD1 with respect to NBD2 results from its position in the molecule or is related to its primary sequence, we constructed and expressed chimeric CFTRs wherein NBD domains were either exchanged or deleted. Synthesis, maturation and activity of the chimeras were assessed by Western blotting and iodide efflux assay after transient or stable expression in COS-1 or CHO cells respectively. The data showed that deletion of NBD1 prevented transport of CFTR to the cytoplasmic membrane whereas deletion of NBD2 did not impair this process but resulted in an inactive chloride channel. On the other hand, substituting or inverting NBDs in the CFTR molecule impaired its processing. In addition, while the NBD1 R555K mutation is known to partially correct the processing of CFTR DeltaF508 and to increase activity of both wild-type and DeltaF508 individual channels, it showed no positive effect when introduced into the double NBD1 chimera. Taken together, these observations suggest that the proper folding process of CFTR results from complex interactions between NBDs and their surrounding domains (MSDs and/or R domain). (+info)
Sodium/iodide symporter: a key transport system in thyroid cancer cell metabolism.
(20/714)
The recent cloning of the gene encoding the sodium/iodide symporter (NIS) has enabled better characterization of the molecular mechanisms underlying iodide transport, thus opening the way to clarifying its role in thyroid diseases. Several studies, at both the mRNA and the protein expression levels, have demonstrated that TSH, the primary regulator of iodide uptake, upregulates NIS gene expression and NIS protein abundance, both in vitro and in vivo. However, other factors, including iodide, retinoic acid, transforming growth factor-beta, interleukin-1alpha and tumour necrosis factor alpha, may participate in the regulation of NIS expression. Investigation of NIS mRNA expression in different thyroid tissues has revealed increased levels of expression in Graves' disease and toxic adenomas, whereas a reduction or loss of NIS transcript was detected in differentiated thyroid carcinomas, despite the expression of other specific thyroid markers. NIS mRNA was also detected in non-thyroid tissues able to concentrate radioiodine, including salivary glands, stomach, thymus and breast. The production of specific antibodies against the NIS has facilitated study of the expression of the symporter protein. Despite of the presence of high levels of human (h)NIS mRNA, normal thyroid glands exhibit a heterogeneous expression of NIS protein, limited to the basolateral membrane of the thyrocytes. By immunohistochemistry, staining of hNIS protein was stronger in Graves' and toxic adenomas and reduced in thyroid carcinomas. Measurement of iodide uptake by thyroid cancer cells is the cornerstone of the follow-up and treatment of patients with thyroid cancer. However, radioiodide uptake is found only in about 67% of patients with persistent or recurrent disease. Several studies have demonstrated a decrease in or a loss of NIS expression in primary human thyroid carcinomas, and immunohistochemical studies have confirmed this considerably decreased expression of the NIS protein in thyroid cancer tissues, suggesting that the low expression of NIS may represent an early abnormality in the pathway of thyroid cell transformation, rather than being a consequence of cancer progression. The relationship between radioiodine uptake and NIS expression by thyroid cancer cells require further study. New strategies, based on manipulation of NIS expression, to obtain NIS gene reactivation or for use as NIS gene therapy in the treatment of radiosensitive cancer, are also being investigated. (+info)
The cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel exhibits lyotropic anion selectivity. Anions that are more readily dehydrated than Cl exhibit permeability ratios (P(S)/P(Cl)) greater than unity and also bind more tightly in the channel. We compared the selectivity of CFTR to that of a synthetic anion-selective membrane [poly(vinyl chloride)-tridodecylmethylammonium chloride; PVC-TDMAC] for which the nature of the physical process that governs the anion-selective response is more readily apparent. The permeability and binding selectivity patterns of CFTR differed only by a multiplicative constant from that of the PVC-TDMAC membrane; and a continuum electrostatic model suggested that both patterns could be understood in terms of the differences in the relative stabilization of anions by water and the polarizable interior of the channel or synthetic membrane. The calculated energies of anion-channel interaction, derived from measurements of either permeability or binding, varied as a linear function of inverse ionic radius (1/r), as expected from a Born-type model of ion charging in a medium characterized by an effective dielectric constant of 19. The model predicts that large anions, like SCN, although they experience weaker interactions (relative to Cl) with water and also with the channel, are more permeant than Cl because anion-water energy is a steeper function of 1/r than is the anion-channel energy. These large anions also bind more tightly for the same reason: the reduced energy of hydration allows the net transfer energy (the well depth) to be more negative. This simple selectivity mechanism that governs permeability and binding acts to optimize the function of CFTR as a Cl filter. Anions that are smaller (more difficult to dehydrate) than Cl are energetically retarded from entering the channel, while the larger (more readily dehydrated) anions are retarded in their passage by "sticking" within the channel. (+info)
Evaluation of three gamma detectors for intraoperative detection of tumors using 111In-labeled radiopharmaceuticals.
(22/714)
Attempts to detect tumors with intraoperative scintillation using tumor-binding radiopharmaceuticals have intensified recently. In some cases previously unknown lesions were found, but in most cases no additional lesions were detected. In this study the physical characteristics of three detector systems and their ability to detect tumors through accumulation of an 111In-labeled radiopharmaceutical were investigated. The first was a sodium iodide (NaI[TI]) detector; the second, a cesium iodide (CsI[TI]) detector; and the third, a cadmium telluride (CdTe) detector. METHODS: A body phantom and tumor phantoms (diameter 5-20 mm) made of water, agarose gel or epoxy with a density and attenuation coefficient similar to those of soft tissue were used to simulate a clinical situation. The activity concentration in the body phantom was based on reported values of 111In-octreotide in normal tissue in humans. The 111In activity concentration in the tumor phantoms varied from 3 to 80 times the 111In activity concentration in the body phantom. Data were processed to determine tumor detection levels. RESULTS: The NaI(TI) detector showed the lowest values for full width at half maximum because this detector had the best collimation, leading to a high ratio between counts from tumor and counts from background, i.e., small tumors could be detected. Because of high efficiency, the CsI(TI) detector sometimes required a somewhat shorter acquisition time to produce a statistically significant difference between tumor phantom and background. For deep-lying tumors the NaI(TI) detector was superior, whereas the CdTe detector was best suited for superficial tumors with a high activity concentration in the underlying tissue. CONCLUSION: At a maximum acquisition time of 30 s, almost all superficial tumors with a diameter of 10 mm or larger were detected if the ratio between the 111In concentration in the tumor and the 111In concentration in the background exceeded 3. However, in clinical situations, biologic variations in the uptake of 111In-octreotide in tumors and in normal tissue makes difficult the determination of a distinct detection level. For such clinical conditions, the NaI(TI) detector is the best choice because it has good resolution despite a lower efficiency. Documentation of detector characteristics is important so that clinicians can make an adequate device in relation to tumor location and receptor expression. (+info)
The HPr kinase from Bacillus subtilis is a homo-oligomeric enzyme which exhibits strong positive cooperativity for nucleotide and fructose 1,6-bisphosphate binding.
(23/714)
Carbon catabolite repression allows bacteria to rapidly alter the expression of catabolic genes in response to the availability of metabolizable carbon sources. In Bacillus subtilis, this phenomenon is controlled by the HPr kinase (HprK) that catalyzes ATP-dependent phosphorylation of either HPr (histidine containing protein) or Crh (catabolite repression HPr) on residue Ser-46. We report here that B. subtilis HprK forms homo-oligomers constituted most likely of eight subunits. Related to this complex structure, the enzyme displays strong positive cooperativity for the binding of its allosteric activator, fructose 1,6-bisphosphate, as evidenced by either kinetics of its phosphorylation activity or the intrinsic fluorescence properties of its unique tryptophan residue, Trp-235. It is further shown that activation of HPr phosphorylation by fructose 1,6-bisphosphate essentially occurs at low ATP and enzyme concentrations. A positive cooperativity was also detected for the binding of natural nucleotides or their 2'(3')-N-methylanthraniloyl derivatives, in either phosphorylation or fluorescence experiments. Most interestingly, quenching of the HprK tryptophan fluorescence by using either iodide or acrylamide revealed a heterogeneity of tryptophan residues within the population of oligomers, suggesting that the enzyme exists in two different conformations. This result suggests a concerted-symmetry model for the catalytic mechanism of positive cooperativity displayed by HprK. (+info)
Gas phase H/D exchange kinetics: DI versus D2O.
(24/714)
The gas phase H/D exchange reactions of bradykinin (M + 3H)3+ ions with D2O and DI were monitored in a quadrupole ion trap mass spectrometer. The H/D exchange kinetics of both chemical probes (D2O and DI) indicate the presence of two noninterconverting reactive gas phase ion populations of bradykinin (M + 3H)3+ at room temperature. The H/D exchange involving DI, however, generally proceeds faster than that involving D2O. The rate observations described here can be rationalized on the basis of the "relay mechanism" (see Campbell et al. J. Am. Chem. Soc. 1995, 117, 12840-12854) recently proposed to account for H/D exchange between D2O and gaseous protonated polypeptides. The higher exchange rate with DI is believed to arise primarily as a result of its lower gas-phase acidity relative to that of D2O and, secondarily, as a result of the longer bond length of DI relative to that of OD in D2O. (+info)