Petr Pancoska

WT p53, but Not Tumor-derived Mutants, Bind to Bcl2 via the DNA Binding Domain and Induce Mitochondrial Permeabilization

The induction of apoptosis by p53 in response to cellular stress is its most conserved function and crucial for p53 tumor suppression. We recently reported that p53 directly induces oligomerization of the BH1,2,3 effector protein Bak, leading to outer mitochondrial membrane permeabilization (OMMP) with release of apoptotic activator proteins. One important mechanism by which p53 achieves OMMP is by forming an inhibitory complex with the anti-apoptotic BclXL protein. In contrast, the p53 complex with the Bcl2 homolog has not been interrogated. Here we have undertaken a detailed characterization of the p53-Bcl2 interaction using structural, biophysical, and mutational analyses. We have identified the p53 DNA binding domain as the binding interface for Bcl2 using solution NMR. The affinity of the p53-Bcl2 complex was determined by surface plasmon resonance analysis (BIAcore) to have a dominant component KD 535 ± 24 nm. Moreover, in contrast to wild type p53, endogenous missense mutants of p53 are unable to form complexes with endogenous Bcl2 in human cancer cells. Functionally, these mutants are all completely or strongly compromised in mediating OMMP, as measured by cytochrome c release from isolated mitochondria. These data implicate p53-Bcl2 complexes in contributing to the direct mitochondrial p53 pathway of apoptosis and further support the notion that the DNA binding domain of p53 is a dual function domain, mediating both its transactivation function and its direct mitochondrial apoptotic function.

Secondary structures comparison of aquaporin-1 and bacteriorhodopsin: a Fourier transform infrared spectroscopy study of two-dimensional membrane crystals

Aquaporins are integral membrane proteins found in diverse animal and plant tissues that mediate the permeability of plasma membranes to water molecules. Projection maps of two-dimensional crystals of aquaporin-1 (AQP1) reconstituted in lipid membranes suggested the presence of six to eight transmembrane helices in the protein. However, data from other sequence and spectroscopic analyses indicate that this protein may adopt a porin-like beta-barrel fold. In this paper, we use Fourier transform infrared spectroscopy to characterize the secondary structure of highly purified native and proteolyzed AQP1 reconstituted in membrane crystalline arrays and compare it to bacteriorhodopsin. For this analysis the fractional secondary structure contents have been determined by using several different algorithms. In addition, a neural network-based evaluation of the Fourier transform infrared spectra in terms of numbers of secondary structure segments and their interconnections [sij] has been performed. The following conclusions were reached: 1) AQP1 is a highly helical protein (42-48% alpha-helix) with little or no beta-sheet content. 2) The alpha-helices have a transmembrane orientation, but are more tilted (21 degrees or 27 degrees, depending on the considered refractive index) than the bacteriorhodopsin helices. 3) The helices in AQP1 undergo limited hydrogen/deuterium exchange and thus are not readily accessible to solvent. Our data support the AQP1 structural model derived from sequence prediction and epitope insertion experiments: AQP1 is a protein with at least six closely associated alpha-helices that span the lipid membrane.

Thermal unfolding of ribonuclease A in phosphate at neutral pH: Deviations from the two–state model

The thermal denaturation of ribonuclease A (RNase A) in the presence of phosphate at neutral pH was studied by differential scanning calorimetry (DSC) and a combination of optical spectroscopic techniques to probe the existence of intermediate states. Fourier transform infrared (FTIR) spectra of the amide I′ band and far‐uv circular dichroism (CD) spectra were used to monitor changes in the secondary structure. Changes in the tertiary structure were monitored by near‐uv CD. Spectral bandshape changes with change in temperature were analyzed using factor analysis. The global unfolding curves obtained from DSC confirmed that structural changes occur in the molecule before the main thermal denaturation transition. The analysis of the far‐uv CD and FTIR spectra showed that these lower temperature–induced modifications occur in the secondary structure. No pretransition changes in the tertiary structure (near‐uv CD) were observed. The initial changes observed in far‐uv CD were attributed to the fraying of the helical segments, which would explain the loss of spectral intensity with almost no modification of spectral bandshape. Separate analyses of different regions of the FTIR amide I′ band indicate that, in addition to α‐helix, part of the pretransitional change also occurs in the β‐strands.

Tumor and liver determinants of prognosis in unresectable hepatocellular carcinoma: A case cohort study

Background and Aims:  A total of 967 patients with unresectable and untransplantable, biopsy‐proven hepatocellular carcinoma (HCC) were prospectively evaluated at baseline and followed up till death.

Novel Use of a Static Modification of Two-Dimensional Correlation Analysis. Part I: Comparison of the Secondary Structure Sensitivity of Electronic Circular Dichroism, FT-IR, and Raman Spectra of Proteins

A modification of Nodas algorithm that allows for calculation of two-dimensional (2D) correlation maps is presented as a method for analysis of a series of (static) spectra of proteins. In this approach, fractional secondary structure was used as the perturbation to generate the 2D correlation. The functional dependence of the spectral intensities on secondary structure is approximated by an even-order polynomial fit to the protein spectra at each spectral frequency. These functions are used to calculate the 2D correlation and disrelation maps, and their regression coefficients are used to weight the results to minimize artifacts. Electronic circular dichroism (ECD), Fourier transform infrared (FT-IR) (amide I and II regions), and Raman spectra of up to 22 proteins are used in the study. Spectral regions identified by the α-helix- and β-sheet-based 2D correlation maps are in agreement with established interpretation of ECD and FT-IR spectra in terms of secondary structure and provide insight into secondary structure assignment for a broad range of Raman bands. Comparison of our functional fit method, specifically designed to identify synchronous correlations, with Nodas Fourier transform-based method, which generates asynchronous maps as well, is discussed.

Thrombocytopenia in relation to tumor size in patients with hepatocellular carcinoma.

Background: Hepatocellular carcinoma (HCC) size at diagnosis is important in management. Without screening programs, tumor size at diagnosis is heterogeneous. Aims: To examine the clinical parameters related to tumor size. Methods: Using prospectively collected data from a 1,100-patient biopsy-proven HCC cohort presenting in the absence of screening, tumor sizes were ordered and trichotomized and the resulting terciles were compared for tumor and blood parameters. Results: The terciles were significantly different with respect to portal hypertension and thrombocytopenia, which were present in a higher percent of tercile I patients with smaller tumors. Tercile III patients with larger HCCs had the highest serum α-fetoprotein (AFP), γ-glutamyl transpeptidase (GGTP), and alkaline phosphatase (ALKP) levels and the most portal vein (PV) thrombosis. Subclassification of tercile I patients by AFP showed that patients with high serum AFP had increased numbers of tumor nodules, more PV thrombosis, higher bilirubin, ALKP, and GGTP levels, and shorter survival. Conclusions: Smaller-tumor tercile I patients had more advanced portal hypertension with thrombocytopenia than did larger-tumor patients. Tercile I patients with higher AFP levels had more frequent PV thrombosis and worse survival than those with lower AFP levels. Elevated serum GGTP and ALKP levels appear to be associated with a more aggressive HCC phenotype. These differing patterns suggest more than one HCC pathway.

Empirical studies of protein secondary structure by vibrational circular dichroism and related techniques. α-Lactalbumin and lysozyme as examples

Vibrational circular dichroism (VCD) has been shown to be sensitive to secondary structure in proteins and peptides and has been used as the basis for quantitative secondary-structure-prediction algorithms. However, the accuracy of these algorithms is not matched by the apparent qualitative sensitivity of the VCD spectra. This report provides examples of the use of VCD to follow structural change spectrally and to clarify the qualitative nature of the structural changes underlying the spectral variation. The VCD spectra and the complementary UV electronic CD (ECD) and FTIR spectra of alpha-lactalbumin (LA) have been studied as a function of pH, denaturation, Ca2+ ion and solvent conditions for several species. Spectral data for lysozyme were compared with those of LA because of their very similar crystal structures. In fact, these proteins in D2O-based pH 7 solution have quite different spectra using these optical techniques. Even for the LA proteins, the human differs from the bovine and goat species. Furthermore, under low pH conditions, where the LAs are in a reversibly denatured, molten globule form, the spectra are more similar, species variation is minimal and the spectral differences from lysozyme are in fact smaller. Our data are consistent with native, pH 7, alpha-lactalbumin having a less well organized structure than lysozyme, possibly in a dynamic sense. Conversely, in the low-pH, molten globule form of LA, tertiary structure is lost which could relax constraints that might distort the helical segments in the native form. The differences between the interpretation of our results and those from X-ray and NMR data may be due to motional sampling of various geometries in LA which all contribute to the spectral signatures seen in optical spectra but whose contributions are washed out in NMR or frozen out in the crystal structure. Part of this flexibility may relate to the rather large 3(10)-helical content in the LA protein structure. Fluctionality may have specific functional effects, perhaps allowing LA to bind better to beta-galactosyl transferase and form the biologically active lactose synthetase complex.

Tumor and liver determinants of prognosis in unresectable hepatocellular carcinoma: a large case cohort study

Objectives967 patients with unresectable and untransplantable, biopsy-proven hepatocellular carcinoma (HCC) were prospectively evaluated at baseline and followed up till death. Survival was the end point.ResultsWe found that male gender, ascites, cirrhosis, portal vein thrombosis (PVT), elevated AFP or bilirubin, or alkaline phosphatase, were each statistically significant adverse prognostic factors. Patients with normal AFP survived longer than those with elevated AFP, even in the presence of PVT, large or bilobar tumors or cirrhosis. We used a bivariate analysis to separate patient sub groups based on liver function and tumor characteristics and found clear discrimination in survival between subsets; in addition both cirrhosis and presence of PVT were significant factors. We also used a purely mathematical approach to derive subgroups and a prognostic model for individual patients. Interestingly, the two approaches gave similar predictive information, which opens the possibility of a more detailed mathematical analysis in the future. The results of this large dataset show that amongst non-surgical HCC patients, there are clear subsets with longer survival.ConclusionThe data supports the concept of heterogeneity of HCC. The three factors, bilirubin, AFP, and PVT predominate in prognosis.

Novel Use of a Static Modification of Two-Dimensional Correlation Analysis. Part II: Hetero-Spectral Correlations of Protein Raman, FT-IR, and Circular Dichroism Spectra

The two-dimensional (2D) static correlation model developed in the previous paper is here extended to hetero-spectral applications for the correlation of spectral features arising from two separate techniques. The calculational method is detailed, and examples are given of Raman vs. circular dichroism (CD) (electronic) and Raman vs. Fourier transform infrared (FT-IR) 2D correlation and disrelation maps with fractional secondary structures used as the perturbation. This approach allows use of well-established secondary structure assignments of spectral features in one technique to interpret structurally less clearly assigned bands, particularly for β-turns, or possibly misassigned bands in other spectra via their calculated 2D correlations.

The influence of carotenoids on the conformation of chlorophyll-protein complexes isolated from the cyanobacterium Plectonema boryanum. Absorption and circular dichroism study

Abstract Thylakoid membranes of the cyanobacterium Plectonema boryanum solubilized with Triton X-100 can be resolved into three fractions of pigment-protein complexes (Hladik, J. and Sofrova, D. (1981) Photosynthetica 15, 490–503). Fraction I contained relatively the highest amount of carotenoids as well as monomeric forms of chlorophyll a, Fractions II and III contained chlorophyll-protein complexes with a characteristic exciton-split circular dichroism in the red region. It has been shown that fraction III is an oligomeric form of the chlorophyll-protein complex of fraction II. Circular dichroism spectra indicate that, different from fraction II, fraction III contains specifically oriented and space-fixed molecules of carotenoids. Thermal dissociation of fracion III to fraction II is accompanied by disappearance of the positive circular dichroism effect of carotenoids in the 500–550 nm region, thus causing deorganization of the carotenoids, proceeding in parallel to the geometrical rearrangement of chlorophyll molecules. Extraction of the carotenoids of fraction III with heptane is acompanied by dissociation of fraction III. We assume that the observed effects are due to binding of the two pigments to the protein component of the complex and that carotenoids can mediate a part of the interactions which stabilize the structure of pigment-protein complexes. Thus, besides the light-harvesting and protective functions, carotenoids can also play a structural role.