RESUMO
The orientation of the ELC region of myosin in skeletal muscle was determined by polarized fluorescence from ELC mutants in which pairs of introduced cysteines were cross-linked by BSR. The purified ELC-BSRs were exchanged for native ELC in demembranated fibers from rabbit psoas muscle using a trifluoperazine-based protocol that preserved fiber function. In the absence of MgATP (in rigor) the ELC orientation distribution was narrow; in terms of crystallographic structures of the myosin head, the LCD long axis linking heavy-chain residues 707 and 843 makes an angle (beta) of 120-125 degrees with the filament axis. This is approximately 30 degrees larger than the broader distribution determined previously from RLC probes, suggesting that, relative to crystallographic structures, the LCD is bent between its ELC and RLC regions in rigor muscle. The ELC orientation distribution in relaxed muscle had two broad peaks with beta approximately 70 degrees and approximately 110 degrees, which may correspond to the two head regions of each myosin molecule, in contrast with the single broad distribution of the RLC region in relaxed muscle. During isometric contraction the ELC orientation distribution peaked at beta approximately 105 degrees , similar to that determined previously for the RLC region.
Assuntos
Rigidez Muscular/metabolismo , Músculos/metabolismo , Cadeias Leves de Miosina/química , Animais , Galinhas , Reagentes de Ligações Cruzadas/farmacologia , Microscopia Confocal , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculos/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , CoelhosRESUMO
As an initial screen for novel markers of renal cancer and to minimise background heterogeneity, we have compared the within-patient profiles of serum samples from seven patients pre- and post-nephrectomy. Samples were depleted of six of the most abundant proteins using Agilent's multiple affinity removal system (MARS) followed by solution-phase IEF prior to separation by 2-DE using narrow range IPG Strips, with a total of 84 gels. The reproducibility of the various steps was demonstrated and an approximate two-fold increase (from 374 to 779) in the number of protein spots observed in the pH region 4.6-7.0 was obtained. However, the majority of additional proteins seen were further isoforms of existing proteins due to the higher resolution and the majority of protein spots identified were still moderate to highly abundant species. Only one protein spot (as yet unidentified) was found to change significantly in the same direction in at least four patients. Although this powerful prefractionation and analysis strategy allows the visualisation of multiple protein isoforms, it is insufficient to allow detection of lower abundance proteins in serum without the implementation of further strategies.
Assuntos
Biomarcadores Tumorais/sangue , Carcinoma de Células Renais/sangue , Fracionamento Químico/métodos , Eletroforese em Gel Bidimensional/métodos , Focalização Isoelétrica/métodos , Neoplasias Renais/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Cromatografia de Afinidade , Feminino , Humanos , Ponto Isoelétrico , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/análise , Nefrectomia , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
PURPOSE: The primary purpose of this study was to determine whether mutations of the class I beta-tubulin gene may be implicated in the inherent resistance to tubulin-binding agents (TBA) in renal cancer, with a small number of samples and cell lines also being examined for class I and III beta-tubulin isotype protein expression. EXPERIMENTAL DESIGN: DNA was extracted from 90 renal tumors and the class I beta-tubulin gene analyzed for mutations. For each sample, eight PCRs were used to cover the complete coding sequence with intronic primers ensuring highly homologous pseudogenes were not coamplified. Additionally, expression levels of class I and III beta-tubulin isotypes in 17 matched normal and malignant renal samples and a panel of renal cell carcinoma cell lines with differing intrinsic resistance to the TBAs was examined by Western blotting. RESULTS: Four polymorphic sequence changes of the class I beta-tubulin gene were identified with no mutations. Class I protein expression levels were higher in tumor tissue versus normal tissue, whereas class III expression showed no consistent change. In renal cancer cell lines, a significant correlation between class III isotype expression and vinblastine sensitivity was observed. CONCLUSIONS: These results do not support a role for mutations in the class I beta-tubulin gene in the intrinsic resistance of renal cancer to TBAs. Class III isotype expression may be implicated in resistance in vitro but in vivo, changes in class I isotype expression in renal cell carcinoma tissue may support a role in resistance to the TBAs and warrants further investigation.
Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Renais/genética , Neoplasias Renais/genética , Tubulina (Proteína)/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/metabolismo , Western Blotting , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Análise Mutacional de DNA , DNA de Neoplasias/química , DNA de Neoplasias/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
Structural changes in myosin power many types of cell motility including muscle contraction. Tilting of the myosin light chain domain (LCD) seems to be the final step in transducing the energy of ATP hydrolysis, amplifying small structural changes near the ATP binding site into nanometer-scale motions of the filaments. Here we used polarized fluorescence measurements from bifunctional rhodamine probes attached at known orientations in the LCD to describe the distribution of orientations of the LCD in active contraction and rigor. We applied rapid length steps to perturb the orientations of the population of myosin heads that are attached to actin, and thereby characterized the motions of these force-bearing myosin heads. During active contraction, this population is a small fraction of the total. When the filaments slide in the shortening direction in active contraction, the long axis of LCD tilts towards its nucleotide-free orientation with no significant twisting around this axis. In contrast, filament sliding in rigor produces coordinated tilting and twisting motions.
Assuntos
Proteínas Motores Moleculares/química , Fibras Musculares Esqueléticas/química , Cadeias Leves de Miosina/química , Animais , Galinhas , Cisteína , Polarização de Fluorescência , Contração Muscular , Fibras Musculares Esqueléticas/fisiologia , Cadeias Leves de Miosina/fisiologia , Estrutura Terciária de ProteínaRESUMO
Although proteomic technologies have been enthusiastically embraced in the field of biomarker discovery and particularly with biological fluids, it is increasingly being recognized that pre-analytical effects, i.e. those occurring prior to the point of actual sample analysis and including factors such as sample processing and storage, can exert marked influences on the results obtained. Such effects have been recognized already for specific analytes in clinical chemistry, but with the increasing sensitivity and resolution of the newer technologies, such effects are potentially even more marked. The challenge of translating initial findings into clinical application requires such issues to be addressed at a very early stage in study design and this paper reviews the current knowledge of the potential impact of pre-analytical factors on proteomic studies of biological fluids.
RESUMO
A primary objective of many protein expression studies is to define expression patterns that can distinguish between normal and diseased states, enabling a better understanding of molecular events associated with disease development and progression and ultimately potentially finding novel markers or therapeutic targets. Exploration and confirmation of many proteins is often done using Western blotting with normalization against "housekeeping proteins", such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin, or beta-tubulin, to correct for protein loading and factors, such as transfer efficiency. Increasingly, in studies examining gene transcript levels, it has been shown that some of the commonly used housekeeping genes may be unsuitable due to the influence of various physiological and pathological factors on their expression. This has not been examined to any great extent for proteins, however. This study examines the degree of variability of three commonly used "housekeeping" proteins (GAPDH, beta-actin, and beta-tubulin) together with class I beta-tubulin, with comparisons being made between a number of different established renal cancer cell lines, matched pairs of renal tumor and normal kidney lysates as well as nine different human tissues and highlights some of the problems encountered.
Assuntos
Carcinoma de Células Renais/metabolismo , Neoplasias Renais/metabolismo , Rim/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas/metabolismo , Actinas/metabolismo , Western Blotting , Carcinoma de Células Renais/química , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Eletroforese em Gel de Poliacrilamida , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Rim/química , Neoplasias Renais/química , Neoplasias Renais/patologia , Proteínas de Neoplasias/química , Proteínas/química , Padrões de Referência , Tubulina (Proteína)/metabolismoRESUMO
Renal cancer is one of the most chemoresistant tumor types. Using a panel of 10 established renal cancer cell lines that have not been subjected to prior drug selection, the range of functional resistance phenotypes to the tubulin-binding agents paclitaxel, vinblastine, vincristine and patupilone (epothilone B, EPO906) was determined, together with expression of P-glycoprotein (PgP), multidrug resistance associated protein-2 (MRP2) and major vault protein (MVP) proteins. The IC(50) values for vincristine correlated positively with PgP expression (r = 0.73; p = 0.031), with values for paclitaxel and vinblastine just failing to reach significance. A significant positive correlation was observed for sensitivity to paclitaxel and MRP2 expression only (r = 0.8; p = 0.013). MVP expression did not correlate with sensitivity to any of the drugs examined. All cell lines exhibited much greater sensitivity to patupilone, demonstrating for the first time the potential use of patupilone in this cancer. In tissue samples from chemotherapy-naive renal cell carcinoma (RCC) patients, marked downregulation or absence of PgP in many tumor cells with expression levels more similar to sensitive cell lines rather than the resistant lines was seen. Similarly, MRP2 was absent or only weakly present in tumor cells, whereas MVP was very strongly upregulated in most tumor samples. This study illustrating discrepancies between results exclusively based on studies in cell lines and findings in vivo suggests that the role of PgP and MRP2 in intrinsic resistance in RCC in vivo may be less than expected from the in vitro findings and supports a potential role for MVP on the basis of in vivo expression studies.
Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/metabolismo , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/metabolismo , Tubulina (Proteína)/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Regulação para Baixo , Humanos , Imuno-Histoquímica , Concentração Inibidora 50 , Rim/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Paclitaxel/química , Fenótipo , Sensibilidade e Especificidade , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologia , Fatores de Tempo , Regulação para Cima , Vimblastina/farmacologia , Vincristina/farmacologiaRESUMO
The orientation of the regulatory light chain (RLC) region of the myosin heads in relaxed skinned fibers from rabbit psoas muscle was investigated by polarized fluorescence from bifunctional rhodamine (BR) probes cross-linking pairs of cysteine residues introduced into the RLC. Pure 1:1 BR-RLC complexes were exchanged into single muscle fibers in EDTA rigor solution for 30 min at 30 degrees C; approximately 60% of the native RLC was removed and stoichiometrically replaced by BR-RLC, and >85% of the BR-RLC was located in the sarcomeric A-bands. The second- and fourth-rank order parameters of the orientation distributions of BR dipoles linking RLC cysteine pairs 100-108, 100-113, 108-113, and 104-115 were calculated from polarized fluorescence intensities, and used to determine the smoothest RLC orientation distribution-the maximum entropy distribution-consistent with the polarized fluorescence data. Maximum entropy distributions in relaxed muscle were relatively broad. At the peak of the distribution, the "lever" axis, linking Cys707 and Lys843 of the myosin heavy chain, was at 70-80 degrees to the fiber axis, and the "hook" helix (Pro830-Lys843) was almost coplanar with the fiber and lever axes. The temperature and ionic strength of the relaxing solution had small but reproducible effects on the orientation of the RLC region.
Assuntos
Modelos Moleculares , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Cadeias Leves de Miosina/fisiologia , Rodaminas/química , Animais , Galinhas , Polarização de Fluorescência/métodos , Fibras Musculares Esqueléticas/química , Cadeias Leves de Miosina/química , Miosinas/química , Miosinas/fisiologia , Coelhos , Sarcômeros/química , Sarcômeros/fisiologiaRESUMO
The structure of the calcium-saturated regulatory domain of skeletal troponin C (sNTnC) complexed with the switch peptide comprising residues 115-131 of troponin I (TnI), and with a bifunctional rhodamine fluorescent label attached to residues 56 (E56C) and 63 (E63C) on the C helix of sNTnC, has been determined using nuclear magnetic resonance (NMR) spectroscopy. The structure shows that the integrity of the C helix is not altered by the E(56,63)C mutations or by the presence of the bifunctional rhodamine and that the label does not interact with the hydrophobic cleft of sNTnC. Moreover, the overall fold of the protein and the position of the TnI peptide are similar to those observed previously with related cardiac NTnC complexes with residues 147-163 of cardiac TnI [Li et al. (1999) Biochemistry 38, 8289-8298] and including the drug bepridil [Wang et al. (2002) J. Biol. Chem. 277, 31124-31133]. The degree of opening of the structure is reduced as compared to that of calcium-saturated sNTnC in the absence of the switch peptide [Gagné et al. (1995) Nat. Struct. Biol. 2, 784-789]. The switch peptide is bound in a shallow and complementary hydrophobic surface cleft largely defined by helices A and B and also has key ionic interactions with sNTnC. These results show that bifunctional rhodamine probes can be attached to surface helices via suitable pairs of solvent-accessible residues that have been mutated to cysteines, without altering the conformation of the labeled domain. A set of such probes can be used to determine the orientation and motion of the target domain in the cellular environment [Corrie et al. (1999) Nature 400, 425-430; Ferguson et al. (2003) Mol. Cell 11(4), in press].
Assuntos
Espectroscopia de Ressonância Magnética , Fibras Musculares Esqueléticas/metabolismo , Troponina C/química , Troponina I/metabolismo , Animais , Cálcio/metabolismo , Galinhas/genética , Galinhas/metabolismo , Dimerização , Hibridização in Situ Fluorescente , Técnicas In Vitro , Fibras Musculares Esqueléticas/química , Mutação , Concentração Osmolar , Isoformas de Proteínas , Estrutura Terciária de Proteína/genética , Rodaminas/metabolismo , Troponina C/genética , Troponina C/metabolismoRESUMO
A recently developed approach for mapping protein-domain orientations in the cellular environment was used to investigate the Ca(2+)-dependent structural changes in the tropomyosin/troponin complex on the actin filament that regulate muscle contraction. Polarized fluorescence from bifunctional rhodamine probes attached along four alpha helices of troponin C (TnC) was measured in permeabilized skeletal muscle fibers. In relaxed muscle, the N-terminal lobe of TnC is less closed than in crystal structures of the Ca(2+)-free domain, and its D helix is approximately perpendicular to the actin filament. In contrast to crystal structures of isolated TnC, the D and E helices are not collinear. On muscle activation, the N lobe orientation becomes more disordered and the average angle between the C helix and the filament changes by 32 degrees +/- 5 degrees. These results illustrate the potential of in situ measurements of helix and domain orientations for elucidating structure-function relations in native macromolecular complexes.