Insights into the intramolecular coupling between the N- and C-domains of troponin C derived from high-pressure, fluorescence, nuclear magnetic resonance, and small-angle X-ray scattering studies.

Troponin C (TnC), the Ca(2+)-binding component of the troponin complex of vertebrate skeletal muscle, consists of two structurally homologous domains, the N- and C-domains; these domains are connected by an exposed α-helix. Mutants of full-length TnC and of its isolated domains have been constructed using site-directed mutagenesis to replace different Phe residues with Trp. Previous studies utilizing these mutants and high hydrostatic pressure have shown that the apo form of the C-domain is less stable than the N-domain and that the N-domain has no effect on the stability of the C-domain [Rocha, C. B., Suarez, M. C., Yu, A., Ballard, L., Sorenson, M. M., Foguel, D., and Silva, J. L. (2008) Biochemistry 47, 5047-5058]. Here, we analyzed the stability of full-length F29W TnC using structural approaches under conditions of added urea and hydrostatic pressure denaturation; F29W TnC is a fluorescent mutant, in which Phe 29, located in the N-domain, was replaced with Trp. From these experiments, we calculated the thermodynamic parameters (ΔV and ΔG°(atm)) that govern the folding of the intact F29W TnC in the absence or presence of Ca(2+). We found that the C-domain has only a small effect on the structure of the N-domain in the absence of Ca(2+). However, using fluorescence spectroscopy, we demonstrated a significant decrease in the stability of the N-domain in the Ca(2+)-bound state (i.e., when Ca(2+) was also bound to sites III and IV of the C-domain). An accompanying decrease in the thermodynamic stability of the N-domain generated a reduction in ΔΔG°(atm) in absolute terms, and Ca(2+) binding affects the Ca(2+) affinity of the N-domain in full-length TnC. Cross-talk between the C- and N-domains may be mediated by the central helix, which has a smaller volume and likely greater rigidity and stability following binding of Ca(2+) to the EF-hand sites, as determined by our construction of low-resolution three-dimensional models from the small-angle X-ray scattering data.

Free-energy linkage between folding and calcium binding in EF-hand proteins.

Troponin is the singular Ca(2+)-sensitive protein in the contraction of vertebrate striated muscles. Troponin C (TnC), the Ca(2+)-binding subunit of the troponin complex, has two distinct domains, C and N, which have different properties despite their extensive structural homology. In this work, we analyzed the thermodynamic stability of the isolated N-domain of TnC using a fluorescent mutant with Phe 29 replaced by Trp (F29W/N-domain, residues 1-90). The complete unfolding of the N-domain of TnC in the absence or presence of Ca(2+) was achieved by combining high hydrostatic pressure and urea, a maneuver that allowed us to calculate the thermodynamic parameters (DeltaV and DeltaG(atm)). In this study, we propose that part of the affinity for Ca(2+) is contributed by the free-energy change of folding of the N- and C-domains that takes place when Ca(2+) binds. The importance of the free-energy change for the structural and regulatory functions of the TnC isolated domains was evaluated. Our results shed light on how the coupling between folding and ion binding contributes to the fine adjustment of the affinity for Ca(2+) in EF-hand proteins, which is crucial to function.

Volume and free energy of folding for troponin C C-domain: linkage to ion binding and N-domain interaction.

Troponin C (TnC) is an 18-kDa acidic protein of the EF-hand family that serves as the trigger for muscle contraction. In this study, we investigated the thermodynamic stability of the C-domain of TnC in all its occupancy states (apo, Mg (2+)-, and Ca (2+)-bound states) using a fluorescent mutant with Phe 105 replaced by Trp (F105W/C-domain, residues 88-162) and (1)H NMR spectroscopy. High hydrostatic pressure was employed as a perturbing agent, in combination with urea or without it. On the basis of changes in Trp emission, the C-domain apo state was denatured by pressure (in the range of 1-1000 bar) in the absence of urea. The fluorescence data were corroborated by following the changes in the (1)H NMR signal of Histidine 128. Addition of Ca (2+) or Mg (2+) increased the C-domain stability so that complete denaturation was attained only by the combined use of high hydrostatic pressure and either 7-8 M or 1.5-2 M urea, respectively. The (1)H NMR spectra in the presence of Ca (2+) was typical of a highly structured protein and allowed us to follow the changes in the local environment of several amino-acid residues as a function of pressure at 4 M Urea. Different residues presented different volume changes, but those that are in the hydrophobic core portrayed values very similar to that obtained for tryptophan 105 as measured by fluorescence, indicating that it is indeed a good probe for the overall tertiary structure. From these experiments, we calculated the thermodynamic parameters (Delta G degrees atm and Delta V) that govern the folding of the C-domain in all its possible physiological states and constructed a thermodynamic cycle. Furthermore, a comparison of the volume and free-energy changes of folding of isolated C-domain with those of intact TnC (F105W) revealed that the N-domain has little effect on the structure of the C-domain, even in the presence of Ca (2+). The volume and free-energy diagrams reveal a landscape of different conformations from the less structured, denatured apo form to the highly structured, Ca (2+)-bound form. The large change in folding free energy of the C-domain that takes place when Ca (2+) binds may explain the much higher Ca (2+) affinity of sites III and IV, 2 orders of magnitude higher than the affinity of sites I and II.

Fourier transform infrared spectroscopy provides a fingerprint for the tetramer and for the aggregates of transthyretin.

Transthyretin (TTR) is an amyloidogenic protein whose aggregation is responsible for several familial amyloid diseases. Here, we use FTIR to describe the secondary structural changes that take place when wt TTR undergoes heat- or high-pressure-induced denaturation, as well as fibril formation. Upon thermal denaturation, TTR loses part of its intramolecular beta-sheet structure followed by an increase in nonnative, probably antiparallel beta-sheet contacts (bands at 1,616 and 1,686 cm(-1)) and in the light scattering, suggesting its aggregation. Pressure-induced denaturation studies show that even at very elevated pressures (12 kbar), TTR loses only part of its beta-sheet structure, suggesting that pressure leads to a partially unfolded species. On comparing the FTIR spectrum of the TTR amyloid fibril produced at atmospheric pressure upon acidification (pH 4.4) with the one presented by the native tetramer, we find that the content of beta-sheets does not change much upon fibrillization; however, the alignment of beta-sheets is altered, resulting in the formation of distinct beta-sheet contacts (band at 1,625 cm(-1)). The random-coil content also decreases in going from tetramers to fibrils. This means that, although part of the tertiary- and secondary-structure content of the TTR monomers has to be lost before fibril formation, as previously suggested, there must be a subsequent reorganization of part of the random-coil structure into a well-organized structure compatible with the amyloid fibril, as well as a readjustment of the alignment of the beta-sheets. Interestingly, the infrared spectrum of the protein recovered from a cycle of compression-decompression at pD 5, 37 degrees C, is quite similar to that of fibrils produced at atmospheric pressure (pH 4.4), which suggests that high hydrostatic pressure converts the tetramers of TTR into an amyloidogenic conformation.

Ca(2+) and Mg(2+) binding to weak sites of TnC C-domain induces exposure of a large hydrophobic surface that leads to loss of TnC from the thin filament.

The C-domain of troponin C, the Ca(2+)-binding subunit of the troponin complex, has two high-affinity sites for Ca(2+) that also bind Mg(2+) (Ca(2+)/Mg(2+) sites), whereas the N-domain has two low-affinity sites for Ca(2+). Two more sites that bind Mg(2+) with very low affinity (K(a)<10(3)M(-1)) have been detected by several laboratories but have not been localized or studied in any detail. Here we investigated the effects of Ca(2+) and Mg(2+) binding to isolated C-domain, focusing primarily on low-affinity sites. Since TnC has no Trp residues, we utilized a mutant with Phe 154 replaced by Trp (F154W/C-domain). As expected from previous reports, the changes in Trp fluorescence revealed different conformations induced by the addition of Ca(2+) or Mg(2+) (Ca(2+)/Mg(2+) sites). Exposure of hydrophobic surfaces of F154W/C-domain was monitored using the fluorescence intensity of bis-anilino naphthalene sulfonic acid. Unlike the changes reported by Trp, the increments in bis-ANS fluorescence were much greater (4.2-fold) when Ca(2+)+Mg(2+) were both present or when Ca(2+) was present at high concentration. Bis-ANS fluorescence increased as a function of [Ca(2+)] in two well-defined steps: one at low [Ca(2+)], consistent with the Ca(2+)/Mg(2+) sites (K(a) approximately 1.5 x 10(6)M(-1)), and one of much lower affinity (K(a) approximately 52.3M(-1)). Controls were performed to rule out artifacts due to aggregation, high ionic strength and formation of the bis-ANS-TnC complex itself. With a low concentration of Ca(2+) (0.6mM) to occupy the Ca(2+)/Mg(2+) sites, a large increase in bis-ANS binding also occurred as Mg(2+) occupied a class of low-affinity sites (K(a) approximately 59 M(-1)). In skinned fibers, a high concentration of Mg(2+) (10-44 mM) caused TnC to dissociate from the thin filament. These data provide new evidence for a class of weak binding sites for divalent cations. They are located in the C-domain, lead to exposure of a large hydrophobic surface, and destabilize the binding of TnC to the regulatory complex even when sites III and IV are occupied.

Interrelationship between bone turnover markers and dietary calcium intake in pregnant women: a longitudinal study.

This longitudinal study evaluated bone turnover and the interrelationship between changes in bone biomarkers and habitual dietary calcium intake during pregnancy in a group of women ranging widely with regard to dietary calcium intake. Thirty-nine healthy pregnant and 30 nonpregnant women were studied. Calcium, phosphorus, 1alpha,25-dihydroxyvitamin D (1,25diHOD), bone alkaline phosphatase (bALP), carboxyterminal propeptides of type I procollagen (PICP) and carboxyterminal telopeptides of type I collagen (betaCTX and ICTP) were measured in serum and calcium, and creatinine and aminoterminal telopeptide (NTX) were determined in urine. Serum calcium and phosphorus did not change but the urinary Ca/Creat ratio and 1,25diHOD increased throughout pregnancy (P < 0.001 and P < 0.0001, respectively). Serum b-ALP and PICP increased during the last two trimesters (P < 0.0001 and P < 0.001, respectively). All studied bone resorption markers increased compared to nonpregnant values throughout pregnancy. The highest increment was observed in the third trimester. The level of significance decreased as follows: betaCTX > NTX >ICTP. Serum 1,25 diHOD versus calcium intake showed a positive and significant correlation (r = 0.51, P < 0.02). A negative correlation between the absolute change in betaCTX, NTX, and b-ALP between the third and second trimester and calcium intake at the end of pregnancy was observed in pregnant women who did not cover adequately calcium intake requirements (r = -0.47, P < 0.03; r = -0.41, P < 0.05; and r = -0.43, P < 0.05, respectively). These results suggest that skeletal response to pregnancy may not be entirely independent of maternal calcium intake, especially in women with usually low calcium intake. In summary, not only hormonal changes in calcium metabolism that occur during pregnancy but also other considerations, such as low dietary calcium intake, may lead to an increment in the biological activity of the skeleton. Additional studies must be conducted to confirm our findings and to gain a better understanding of skeletal response to a low calcium intake during pregnancy.

Dissociation of amyloid fibrils of alpha-synuclein and transthyretin by pressure reveals their reversible nature and the formation of water-excluded cavities.

Protein misfolding and aggregation have been linked to several human diseases, including Alzheimer's disease, Parkinson's disease, and systemic amyloidosis, by mechanisms that are not yet completely understood. The hallmark of most of these diseases is the formation of highly ordered and beta-sheet-rich aggregates referred to as amyloid fibrils. Fibril formation by WT transthyretin (TTR) or TTR variants has been linked to the etiology of systemic amyloidosis and familial amyloid polyneuropathy, respectively. Similarly, amyloid fibril formation by alpha-synuclein (alpha-syn) has been linked to neurodegeneration in Parkinson's disease, a movement disorder characterized by selective degeneration of dopaminergic neurons in the substantia nigra. Here we show that consecutive cycles of compression-decompression under aggregating conditions lead to reversible dissociation of TTR and alpha-syn fibrils. The high sensitivity of amyloid fibrils toward high hydrostatic pressure (HHP) indicates the existence of packing defects in the fibril core. In addition, through the use of HHP we are able to detect differences in stability between fibrils formed from WT TTR and the familial amyloidotic polyneuropathy-associated variant V30M. The fibrils formed by WT alpha-syn were less susceptible to pressure denaturation than the Parkinson's disease-linked variants, A30P and A53T. This finding implies that fibrils of alpha-syn formed from the variants would be more easily dissolved into small oligomers by the cellular machinery. This result has physiological importance in light of the current view that the pathogenic species are the small aggregates rather the mature fibrils. Finally, the HHP-induced formation of fibrils from TTR is relatively fast (approximately 60 min), a quality that allows screening of antiamyloidogenic drugs.

Role of hydration in the closed-to-open transition involved in Ca2+ binding by troponin C.

Troponin C (TnC) is the Ca(2+)-binding subunit of the troponin complex of vertebrate skeletal muscle. It consists of two structurally homologous domains, N and C, connected by an exposed alpha-helix. The C-domain has two high-affinity sites for Ca(2+) that also bind Mg(2+), whereas the N-domain has two low-affinity sites for Ca(2+). Previous studies using isolated N- and C-domains showed that the C-domain apo form was less stable than the N-domain. Here we analyzed the stability of isolated N-domain (F29W/N-domain) against urea and pressure denaturation in the absence and in the presence of glycerol using fluorescence spectroscopy. Increasing the glycerol concentration promoted an increase in the stability of the protein to urea (0-8 M) in the absence of Ca(2+). Furthermore, the ability to expose hydrophobic surfaces normally promoted by Ca(2+) binding or low temperature under pressure was partially lost in the presence of 20% (v/v) glycerol. Glycerol also led to a decrease in the Ca(2+) affinity of the N-domain in solution. From the ln K(obs) versus ln a(H)2(O), we obtained the number of water molecules (63.5 +/- 8.7) involved in the transition N <=>N:Ca(2) that corresponds to an increase in the exposed surface area of 571.5 +/- 78.3 A(2). In skinned fibers, the affinity for Ca(2+) was also reduced by glycerol, although the effect was much less pronounced than in solution. Our results demonstrate quantitatively that the stability of this protein and its affinity for Ca(2+) are critically dependent on protein hydration.

Carcinoma epidermôide primário da glândula mamária / Primary squamous cell carcinoma of the breast

Os autores apresentam um caso extremamente raro de carcinoma espinocelular primário da glândula mamária direita. Tratava-se de uma paciente de 41 anos, com antecedentes cirúrgicos há 1 ano e meio de papiloma intraductal e mastite crônica, que referia lesäo tipo abscesso, que drenava material hemopurulento. O processo piorou, e apalpou-se nódulo profundo, cujo diagnóstico anátomo-patológico após cirurgia foi carcinoma epidermóide bem diferenciado. Apesar do tratamento agressivo, a evoluçäo foi rapidamente mortal. A histogênese, assim como os diagnósticos diferenciados, é discutida

Progressive premalignant and malignant development (metachronous appearance) in multiple burn scars (Marjolin's ulcers): report of a rare case and review of the literature

O epônimo úlcera de Marjolin refere-se à transformação maligna de qualquer úlcera ou cicatriz da pele. Relata-se o caso de uma mulher, branca, de 35 anos de idade, que sofreu queimadura térmica na metade inferior do seu corpo quando estava com 8 anos de idade. Após 16 anos, e ao longo de 9 anos, apareceram em forma metácrona na pele de ambas as coxas, úlceras, lesões de hiperplasia pseudocarcinomatosa e carcinomas epidermóides. Discute-se a história clínica, a histopatologia e histogênese, o comportamento biológico, assim como o tratamento. A literatura mais relevante foi revisada.

Tumores carcinóides mucinosos da glândula mamária / Mucinous carcinoid tumors of the breast

Na avaliaçäo retrospectiva de 28 pacientes lobulares mamários, os autores encontram três casos de carcinoma neuroendócrino com diferenciaçäo mucinosa (também conhecidos como carcinóide mucossecretante). Tratavam-se de mulheres jovens (média de 40,6 anos) com tumores nos quadrantes laterais da glândula mamária direita. Os tumores tinham aspecto sólido ou cístico-gelatinoso, diâmetro médio de 30 mm e margens bem delimitadas. Na histopatologia, o padräo basalóide foi o mais freqüente, com acúmulo de células sem atipias nem mitoses, apresentando argirofilia positiva, além da presença de mucina extracelular em porcentagens variável, nunca superior a 33 por cento. Nenhum dos casos, apresentou metástases axilares nem à distância, e näo foi observada síndrome carcinóide. Discute-se a histogênese, o comportamento biológico e o tratamento dessa neoplasia

Tumor carcinóide neuroendócrino da mama: estudo clínico-patológico de seis casos / Neuroendocrine carcinoid tumors of breast: clinicopathologic study of six cases

O tumor carcinóide primário da glândula mamária é uma neoplasia muito rara, de existência controversa. Sua histogênese ainda é discutida, sendo a origem mais reconhecida as células basais primitivas ductais. No estudo retrospectivo de 243 carcinomas infiltrativos e "in situ" de tipo ductal e lobular, seguindo estritos critérios de seleçäo, foram achados seis tumores carcinóides. O material correspondia a mulheres com idade média de 43 anos, que apresentaram-se com massas palpáveis notadamente nos quadrantes externos da glândula mamária direita. Näo foram comprovadas metástases axilares nem síndrome carcinóide. A mamografia mostrou opacidades densas de contornos regulares. A punçäo citológica com agulha fina sugeriu células neoplásicas de tipo lobular em três casos; hiperplasia lobular atípica em um caso e presença de mucina näo específica no resto. No estádio II da doença foi realizada mastectomia de tipo Madden em todos os casos. Macroscopicamente os tumores tinham uma média de 29,8 mm de diâmetro, aspecto sólido em quatro casos e cístico-gelatinoso em dois, e margens bem definidas. O padräo histopatológico mais freqüente foi o basalóide e presença de mucina extracelular compondo 28 porcento do tumor em três casos. Näo foram comprovadas mitoses. Os diagnósticos diferenciais incluíram: o carcinoma lobular, o carcinoma mucinoso e as metástases mamárias de carcinóides de outras topografias. Na evoluçäo pós-operatória näo foi comprovada recorrência local, disseminaçäo a distância ou óbito numa média de 3,8 anos, sendo que em nenhuma das pacientes realizou-se rádio, químio ou hormonioterapia. Baseados nesses dados propöe-se a quadrantectomia com esvaziamento axilar como primeira forma de tratamento, reservando a mastectomia para um segundo momento.