Stefka G. Taneva

Reconstitution of Proapoptotic BAK Function in Liposomes Reveals a Dual Role for Mitochondrial Lipids in the BAK-driven Membrane Permeabilization Process

BAK is a key effector of mitochondrial outer membrane permeabilization (MOMP) whose molecular mechanism of action remains to be fully dissected in intact cells, mainly due to the inherent complexity of the intracellular apoptotic machinery. Here we show that the core features of the BAK-driven MOMP pathway can be reproduced in a highly simplified in vitro system consisting of recombinant human BAK lacking the carboxyl-terminal 21 residues (BAKΔC) and tBID in combination with liposomes bearing an appropriate lipid environment. Using this minimalist reconstituted system we established that tBID suffices to trigger BAKΔC membrane insertion, oligomerization, and pore formation. Furthermore, we demonstrate that tBID-activated BAKΔC permeabilizes the membrane by forming structurally dynamic pores rather than a large proteinaceous channel of fixed size. We also identified two distinct roles played by mitochondrial lipids along the molecular pathway of BAKΔC-induced membrane permeabilization. First, using several independent approaches, we showed that cardiolipin directly interacts with BAKΔC, leading to a localized structural rearrangement in the protein that “primes” BAKΔC for interaction with tBID. Second, we provide evidence that selected curvature-inducing lipids present in mitochondrial membranes specifically modulate the energetic expenditure required to create the BAKΔC pore. Collectively, our results support the notion that BAK functions as a direct effector of MOMP akin to BAX and also adds significantly to the growing evidence indicating that mitochondrial membrane lipids are actively implicated in BCL-2 protein family function.

Microcalorimetry of Blood Serum Proteome: A Modified Interaction Network in the Multiple Myeloma Case

Hereby we report on a novel approach in the study of multiple myeloma (MM), namely, differential scanning calorimetry (DSC) combined with serum protein electrophoresis. Distinct thermodynamic signatures describe the DSC thermograms of MM blood sera, in contrast to the unique profile found for healthy individuals. The thermal behavior of MM sera reflects a complex interplay between the serum concentration and isotype of the M protein and of albumin, and modified ligand- and/or protein-protein interactions, resulting in stabilization of globulins and at least a fraction of albumin. In all MM cases the 85 °C, transferrin-assigned transition is missing. A distinct feature of IgG isotype (κ and λ) DSC profiles only is the presence of a transition at 82 °C. A DSC-based classification of MM depicts two sets of melting patterns (MMt2 and MMt3 with two or three successive thermal transitions), and subsets within each set (MMt2(i) or MMt3(i), the subscript i = 1, 2 or 3 denotes the main transition being one of the three transitions). The results demonstrate the potential of DSC to monitor MM-related modifications of the serum proteome, even at low M protein concentrations, Bence Jones and importantly nonsecretory multiple myeloma cases, and prove DSC as a versatile tool for oncohematology.

Calorimetry-based profiling of blood plasma from colorectal cancer patients

BACKGROUND Differential scanning calorimetry (DSC), a highly sensitive technique for resolving thermally-induced protein folding/unfolding transitions, recently was recognized as a novel tool for disease diagnosis and monitoring. To further elaborate this approach we have applied DSC in a study of blood plasma from patients with colorectal cancer (CRC) at different stages of tumor development and localization. METHODS Blood plasma from patients diagnosed with CRC was analyzed by DSC. The CRC thermograms were compared to those of healthy individuals and patients with gastric cancer and non-cancerous soft tissue inflammation. The thermodynamic parameters: excess heat capacity and enthalpy of the transitions corresponding to the most abundant plasma proteins, as well as transition and first moment temperatures were determined from the calorimetric profiles. RESULTS The calorimetric profiles of blood plasma from CRC patients are found to be distinct from those of healthy individuals and those of patients with soft tissue, non-cancerous inflammation. Generally the CRC thermograms exhibit reduced heat capacity of the major albumin/globulin-assigned thermal transitions, lower enthalpy and a featureless high temperature region compared to healthy individuals. CONCLUSIONS A classification of blood plasma proteome from patients with colorectal cancer (CRC1, CRC2 and CRC3 groups, and subgroups within each group CRC1(1-2), CRC2(1-2) and CRC3(1-2)) is proposed based on the derived thermodynamic parameters. GENERAL SIGNIFICANCE The presented data demonstrate a proof of principle and confirm that the DSC technique has a potential to monitor changes in the CRC blood plasma proteome. This study is a further step toward the validation of calorimetry as a diagnostic tool.

A pathway for the thermal destabilization of bacteriorhodopsin

A variety of structural techniques, including IR spectroscopy, reveals that thermal denaturation of bacteriorhodopsin follows a given pathway (successively rearrangement of helical structures, extensive deuterium exchange, and finally protein aggregation) irrespective of heating rate, pH or ionic strength conditions. In all cases, thermal denaturation leads to a ‘compact denatured state’ which retains a large proportion of ordered structure.

DnaJ Recruits DnaK to Protein Aggregates

Thermal stress might lead to protein aggregation in the cell. Reactivation of protein aggregates depends on Hsp100 and Hsp70 chaperones. We focus in this study on the ability of DnaK, the bacterial representative of the Hsp70 family, to interact with different aggregated model substrates. Our data indicate that DnaK binding to large protein aggregates is mediated by DnaJ, and therefore it depends on its affinity for the cochaperone. Mutations in the structural region of DnaK known as the “latch” decrease the affinity of the chaperone for DnaJ, resulting in a defective activity as protein aggregate-removing agent. As expected, the chaperone activity is recovered when DnaJ concentration is raised to overcome the lower affinity of the mutant for the cochaperone, suggesting that a minimum number of aggregate-bound DnaK molecules is necessary for its efficient reactivation. Our results provide the first experimental evidence of DnaJ-mediated recruiting of ATP-DnaK molecules to the aggregate surface.

Electric properties of thylakoid membranes from pea mutants with modified carotenoid and chlorophyll-protein complex composition

Surface electric properties of thylakoid membranes from wild type and two mutant forms, Coeruleovireus 2/16 and Costata 2/133, of pea are investigated by electric light scattering and microelectrophoretic measurements. Characterization of the chlorophyll–protein complexes in thylakoid membranes reveals that the relative ratio of oligomeric (LHC II1) to monomeric (LHC II3) forms of the light-harvesting Chl a/b complex of Photosystem II is lower (3.34) in 2/133 mutant and higher (6.62) in 2/16 mutant than in wild type (4.57). This is accompanied by elevated amounts and a considerable reduction of all carotenoids in 2/16 and 2/133 mutant, respectively, as compared to the wild type. The concomitant variations of the permanent dipole moment (transversal charge asymmetry), electric polarizability and electrokinetic charge of the thylakoid membranes from both the mutants are discussed in terms of the differences in the supramolecular (oligomeric) organization of the light-harvesting complexes II within the photosynthetic apparatus.

Recognition of nucleoplasmin by its nuclear transport receptor importin α/β: insights into a complete import complex.

Nuclear import of the pentameric histone chaperone nucleoplasmin (NP) is mediated by importin α, which recognizes its nuclear localization sequence (NLS), and importin β, which interacts with α and is in charge of the translocation of the NP/α/β complex through the nuclear pore. Herein, we characterize the assembly of a functional transport complex formed by full-length NP with importin α/β. Isothermal titration calorimetry (ITC) was used to analyze the thermodynamics of the interactions of importin α with β, α with NP, and the α/β heterodimer with NP. Our data show that binding of both importin α and α/β to NP is governed by a favorable enthalpic contribution and that NP can accommodate up to five importin molecules per NP pentamer. Phosphomimicking mutations of NP, which render the protein active in histone chaperoning, do not modulate the interaction with importin. Using small-angle X-ray scattering, we model the α/β heterodimer, NP/α, and NP/α/β solution structures, which reveal a glimpse of a complete nuclear import complex with an oligomeric cargo protein. The set of alternative models, equally well fitting the scattering data, yields asymmetric elongated particles that might represent consecutive geometries the complex can adopt when stepping through the nuclear pore.

Energetics of nucleotide-induced DnaK conformational states.

Hsp70 chaperones are molecular switches that use the free energy of ATP binding and hydrolysis to modulate their affinity for protein substrates and, most likely, to remodel non-native interactions allowing proper substrate folding. By means of isothermal titration calorimetry, we have measured the thermodynamics of ATP and ADP binding to (i) wild-type DnaK, the main bacterial Hsp70; (ii) two single-point mutants, DnaK(T199A), which lacks ATPase activity but maintains conformational changes similar to those observed in the wild-type protein, and DnaK(R151A), defective in interdomain communication; and iii) two deletion mutants, the isolated nucleotide binding domain (K-NBD) and a DeltaLid construct [DnaK(1-507)]. At 25 degrees C, ATP binding to DnaK results in a fast endothermic and a slow exothermic process due to ATP hydrolysis. We demonstrate that the endothermic event is due to the allosteric coupling between ATP binding to the nucleotide binding domain and the conformational rearrangement of the substrate binding domain. The interpretation of our data is compatible with domain docking upon ATP binding and shows that this conformational change carries an energy penalty of ca. 1 kcal/mol. The conformational energy stored in the ATP-bound DnaK state, together with the free energy of ATP hydrolysis, can be used in remodeling bound substrates.

Allosteric Communication between the Nucleotide Binding Domains of Caseinolytic Peptidase B

ClpB is a hexameric chaperone that solubilizes and reactivates protein aggregates in cooperation with the Hsp70/DnaK chaperone system. Each of the identical protein monomers contains two nucleotide binding domains (NBD), whose ATPase activity must be coupled to exert on the substrate the mechanical work required for its reactivation. However, how communication between these sites occurs is at present poorly understood. We have studied herein the affinity of each of the NBDs for nucleotides in WT ClpB and protein variants in which one or both sites are mutated to selectively impair nucleotide binding or hydrolysis. Our data show that the affinity of NBD2 for nucleotides (Kd = 3–7 μm) is significantly higher than that of NBD1. Interestingly, the affinity of NBD1 depends on nucleotide binding to NBD2. Binding of ATP, but not ADP, to NBD2 increases the affinity of NBD1 (the Kd decreases from ≈160–300 to 50–60 μm) for the corresponding nucleotide. Moreover, filling of the NBD2 ring with ATP allows the cooperative binding of this nucleotide and substrates to the NBD1 ring. Data also suggest that a minimum of four subunits cooperate to bind and reactivate two different aggregated protein substrates.

Effect of phosphatidylglycerol depletion on the surface electric properties and the fluorescence emission of thylakoid membranes

To explore the possible effect of phosphatidylglycerol (PG) on the surface electric properties and chlorophyll fluorescence characteristics we used electric light scattering technique and 77K chlorophyll fluorescence of thylakoid membranes from a cyanobacterium, Synechocystis PCC6803 (wild type) and its pgsA mutant defective in PG synthesis. We found a strong decrease in the permanent and induced electric dipole moments of the mutant thylakoids, following long-term PG depletion parallel with a decrease of the emission peak from PSI and an increase of the emission peak from PSII. Partial recovery of the electric state of thylakoid membranes was observed at re-addition of PG to the mutant cells depleted of PG for 21 days. This change in the electric dipole moments is probably due to a decrease in PG content and progressive structural alterations in the macroorganization of the photosynthetic complexes induced by PG deprivation. Our results suggest that the depletion of a lipid, which carries a negative charge, despite its small contribution to the overall lipid content, significantly perturbs the surface charge of the membranes. These changes are related with the chlorophyll fluorescence emission ratios of two photosystems and may partly explain our earlier results concerning the PG requirement for the function and assembly of photosystems I and II reaction centers.