Simulations of reversible protein aggregate and crystal structure.

We simulated the structure of reversible protein aggregates as a function of protein surface characteristics, protein-protein interaction energies, and the entropic penalty accompanying the immobilization of protein in a solid phase. These simulations represent an extension of our previous work on kinetically irreversible protein aggregate structure and are based on an explicit accounting of the specific protein-protein interactions that occur within reversible aggregates and crystals. We considered protein monomers with a mixture of hydrophobic and hydrophilic surface regions suspended in a polar solvent; the energetic driving force for aggregation is provided by the burial of solvent-exposed hydrophobic surface area. We analyzed the physical properties of the generated aggregates, including density, protein-protein contact distributions, solvent accessible surface area, porosity, and order, and compared our results with the protein crystallization literature as well as with the kinetically irreversible case. The physical properties of reversible aggregates were consonant with those observed for the irreversible aggregates, although in general, reversible aggregates were more stable energetically and were more crystal-like in their order content than their irreversible counterparts. The reversible aggregates were less dense than the irreversible aggregates, indicating that the increased energetic stability is derived primarily from the optimality rather than the density of the packing in the solid phase. The extent of hydrophobic protein-protein contacts and solvent-exposed surface area within the aggregate phase depended on the aggregation pathway: reversible aggregates tended to have a greater proportion of hydrophobic-hydrophobic contacts and a smaller fraction of hydrophobic solvent-exposed surface area. Furthermore, the arrangement of hydrophobic patches on the protein surface played a major role in the distribution of protein contacts and solvent content. This was readily reflected in the order of the aggregates: the greater the contiguity of the hydrophobic patches on the monomer surface, the less ordered the aggregates became, despite the opportunities for rearrangement offered by a reversible pathway. These simulations have enhanced our understanding of the impact of protein structural motifs on aggregate properties and on the demarcation between aggregation and crystallization.     

Mechanisms of photodestruction of the eye structure. Formation of polypeptide aggregates upon UV-irradiation of lens proteins

UV-light injury of individual crystallins (water soluble proteins of the cattle eye crystalline lens) were studied by SDS PSSG technique. Photodamage resulted in oligomer formation. The appearance of high molecular aggregates with the molecular mass as large as 10(5) D were seen in all fractions of the crystalline.     

Physiological characteristics of Salmonella typhimurium treated with penicillin]

Growing bacteria of the two strains of Salmonella typhimurium differing in the sensitivity levels to UV-light formed multinuclear non-septal filaments in the penicillin-containing nutrient medium. The maximum number of the lifefull filaments was formed by the 4th hour of incubation in the beaf-peptone broth at a temperature of 37 degrees C in the presence of 5 gamma/ml of penicillin. The strains exposed to penicillin were less sensitive to UV-light. Exclusion of penicillin from the nutrient medium resulted in a new division of the filamentous cells and reduction of the initial UV-light sensitivity level. It was concluded that the low UV-light sensitivity level of the filaments induced by penicillin was associated with their multinuclear state.     

Relation between molecular shape and the morphology of self-assembling aggregates: a simulation study

Proteins can aggregate in a wide variety of structures, both compact and extended. We present simulations of a coarse-grained anisotropic model that reproduce many of the experimentally observed aggregate structures. Conversely, all structures predicted by our model have experimental counterparts (ribbons, multistranded fibrils, and vesicles). The model we use is that of a rodlike particle with an attractive (hydrophobic) stripe on its side. Our Monte Carlo simulations show that aggregate morphologies crucially depend on two parameters. The first one is the width of the attractive stripe and the second one is a presence or absence of attractive interactions at the particle ends. These results provide us with a generic insight into the relation between the shape of protein-protein interaction potential and the morphology of protein aggregates.     

The formation and repair of single-strand breaks in DNA of cultured mammalian cells treated with UV-light, methylating agents or 4-nitroquinoline-1-oxide.

The technique of sedimentation in alkaline sucrose was used to examine the formation and repair of single-strand (SS) breaks in cultured mammalian cells that were treated with methyl methanesulfonate (MMS), methyl nitrosourea (MNUA), 4-nitroquinoline-1-oxide (4NQO) or UV-light. The SS breaks induced by MMS and 4NQO were largely repaired by HeLa cells during a 5-h post-treatment incubation. The SS breaks induced by MNUA and UV-light were not repaired by HeLa cells. L-cells were not able to repair the SS breaks induced by any of the agents, which correlates with the deficiency of these cells for repair synthesis of DNA. The following conclusions are discussed. MNUA and UV-light produce modifications in DNA which are not repaired but are translated into SS breaks in alkali. MMS produces SS breaks intracellularly but these are not derived from a simple depurination of methylated purines. 4NQO produces a modification in DNA which is translated into an SS break in alkali but which can be removed by an intracellular process.     

Structural deformation upon protein-protein interaction: A structural alphabet approach

Background  

In a number of protein-protein complexes, the 3D structures of bound and unbound partners significantly differ, supporting the induced fit hypothesis for protein-protein binding.     

Form-determining functions in Sindbis virus nucleocapsids: nucleosomelike organization of the nucleocapsid.

Purified intact Sindbis virus nucleocapsids were treated at different pH values or with various concentrations of divalent cations, cation chelators, salt, or formamide. The resulting structures were examined by velocity sedimentation, electron microscopy, and protein-protein cross-linking. Changes in each of the test conditions led to alterations in the sedimentation profile of treated nucleocapsids. Appropriate concentrations of formamide or divalent cations generated beaded strandlike structures similar in morphology to those generated from adenovirus cores and nucleosomes. The capsid protein and RNA remained associated with each other at NaCl concentrations less than or equal to 1 M or after treatment of the structures with alkaline pH up to and including pH 10.7. Protein and RNA were dissociated by salt concentrations of greater than 1 M, suggesting that the arginine-rich, amino-terminal portion of the capsid protein is responsible for binding the RNA. Protein-protein cross-linking also indicated that the capsid proteins remained associated in small aggregates under some of the conditions that caused dissociation of the nucleocapsid and suggested the presence of more than one type of protein-protein interaction in the nucleocapsids. Collectively, these data suggest that, like histones and adenovirus core proteins, the Sindbis virus capsid protein serves to package segments of the genome into nucleoprotein beads which are capable of interacting with each other to form the nucleocapsid structure.     

Low-dose UV-radiation sensitizes keratinocytes to TRAIL-induced apoptosis

The impact of low-dose ultraviolet light (UV-light) on apoptotic susceptibility of keratinocytes (KCs) induced by TRAIL is unclear. Skin expresses a functional form of TRAIL, and while sun exposure influences TRAIL death receptors, a role for decoy receptors has not been evaluated. Unraveling mechanisms involving apoptotic sensitivity of KCs is important because skin is the first target of UV-light, and a site for commonly occurring cancers. Since apoptosis is a homeostatic process eliminating UV-light induced DNA damaged cells, elucidating molecular events regulating apoptosis enhances understanding of cutaneous photocarcinogenesis. Here we demonstrate low-dose UV-light enhances susceptibility of KCs to TRAIL-induced apoptosis. Low-dose UV-light selectively reduces decoy receptors, without influencing death receptor levels. UV-induced enhanced apoptotic susceptibility was reduced by over-expression of decoy receptor TRAIL-R4, but not TRAIL-R3; or treatment with thiol compound pyrrolidine dithiocarbamate (PDTC), which also enhanced TRAIL-R4 levels. Besides influencing decoy receptors, low-dose UV-light plus TRAIL also synergistically promoted cytochrome c and Smac release from mitochondria. Inhibitors directed against caspases 2, 3, 8, and 9 reduced the synergistic apoptotic response following low-dose UV-light plus TRAIL exposure; as did forced over-expression of Bcl-x and dominant negative (DN) constructs of FADD and caspase 9. Thus, relative levels of decoy receptors significantly influence susceptibility of KCs to TRAIL-induced apoptosis with concomitant low-dose UV-light exposure; in addition to the apoptotic pathway mediated by mitochondrial permeabilization.     

Protein-protein docking by simulating the process of association subject to biochemical constraints

We present a computational procedure for modeling protein-protein association and predicting the structures of protein-protein complexes. The initial sampling stage is based on an efficient Brownian dynamics algorithm that mimics the physical process of diffusional association. Relevant biochemical data can be directly incorporated as distance constraints at this stage. The docked configurations are then grouped with a hierarchical clustering algorithm into ensembles that represent potential protein-protein encounter complexes. Flexible refinement of selected representative structures is done by molecular dynamics simulation. The protein-protein docking procedure was thoroughly tested on 10 structurally and functionally diverse protein-protein complexes. Starting from X-ray crystal structures of the unbound proteins, in 9 out of 10 cases it yields structures of protein-protein complexes close to those determined experimentally with the percentage of correct contacts >30% and interface backbone RMSD <4 A. Detailed examination of all the docking cases gives insights into important determinants of the performance of the computational approach in modeling protein-protein association and predicting of protein-protein complex structures.     

Mutagenesis in cloned yeast genes. The effect of mutation rad2 on the frequency of gene mutation in plasmid and chromosome

The influence of rad2 mutation blocking incision of pyrimidine dimers on frequency of UV-light and 6-hydroxylaminopurine (6-GAP)-induced adenine-independent revertants was studied in the strains of Saccharomyces cerevisiae containing the same mutant allele of gene ADE2 in episomic plasmid and in chromosome. It was shown that the strains carrying the ade2 mutation in chromosome and in plasmid did not differ in sensitivity to lethal action of UV-light and 6-GAP. However, in the plasmid rad2 strain reversions were induced by UV-light more frequently (approximately 100 times), as compared to the chromosome strain. We observed no significant differences between reversion frequencies in plasmid and chromosome RAD strains. The tendency to enhanced 6-GAP-induced mutagenesis, less sharply expressed, was observed in the chromosome rad2 strain, as compared to the plasmid one. However, the plasmid RAD strain was characteristic of higher reversion frequency induced by 6-GAP, as compared to the chromosome strain. The possible mechanisms of these phenomena are discussed.     

Blue autofluorescence in protein aggregates “lighted on” by UV induced oxidation

Oxidation of amino acid side chains in protein structure can be induced by UV irradiation leading to critical changes in molecular structure possibly modifying protein stability and bioactivity.Here we show, by using a combination of multiple spectroscopic techniques and Fluorescence Lifetime Imaging, that UV-light exposure induces irreversible oxidation processes in Ubiquitin structure. In particular, the growth of a new autofluorescence peak in the blue region is detected, that we attribute to tyrosine oxidation products. Blue autofluorescence intensity is found to progressively increase also during aggregation processes leading to the formation of aggregates of non-amyloid nature.Significantly, analogous spectral modifications are found in amyloid fibrils from human insulin and Amyloid-β peptide grown under UV exposure. Experimental results reveal a substantial overlap between the fluorescence signal here attributed to tyrosine oxidation and the one referred in literature as “Amyloid autofluorescence”. These findings clearly represent a caveat about the specificity of the blue fluorescence peak measured for amyloids, especially when grown in conditions in which tyrosine residues may be oxidized.Moreover, our results once again highlight the close link between the formation of amyloid aggregates and protein damage resulting from oxidative stress, as these neurotoxic aggregate species are found to contain damaged residues.     

Simulations of kinetically irreversible protein aggregate structure.

We have simulated the structure of kinetically irreversible protein aggregates in two-dimensional space using a lattice-based Monte-Carlo routine. Our model specifically accounts for the intermolecular interactions between hydrophobic and hydrophilic protein surfaces and a polar solvent. The simulations provide information about the aggregate density, the types of inter-monomer contacts and solvent content within the aggregates, the type and extent of solvent exposed perimeter, and the short- and long-range order all as a function of (i) the extent of monomer hydrophobic surface area and its distribution on the model protein surface and (ii) the magnitude of the hydrophobic-hydrophobic contact energy. An increase in the extent of monomer hydrophobic surface area resulted in increased aggregate densities with concomitant decreased system free energies. These effects are accompanied by increases in the number of hydrophobic-hydrophobic contacts and decreases in the solvent-exposed hydrophobic surface area of the aggregates. Grouping monomer hydrophobic surfaces in a single contiguous stretch resulted in lower aggregate densities and lower short range order. More favorable hydrophobic-hydrophobic contact energies produced structures with higher densities but the number of unfavorable protein-protein contacts was also observed to increase; greater configurational entropy produced the opposite effect. Properties predicted by our model are in good qualitative agreement with available experimental observations.     

Covalent tethering of the dimer interface annuls aggregation in thymidylate synthase.

Thymidylate synthase (TS), a dimeric enzyme, forms large soluble aggregates at concentrations of urea (3.3-5M), well below that required for complete denaturation, as established by fluorescence and size-exclusion chromatography. In contrast to the wild-type enzyme, an engineered mutant of TS (T155C/E188C/C244T), TSMox, in which two subunits are crosslinked by disulfide bridges between residues 155-188' and 188-155' does not show this behavior. Aggregation behavior is restored upon disulfide bond reduction in the mutant protein, indicating the involvement of interface segments in forming soluble associated species. Intermolecular disulfide crosslinking has been used as a probe to investigate the formation of larger non-native aggregates. The studies argue for the formation of large multimeric species via a sticky patch of polypeptide from the dimer interface region that becomes exposed on partial unfolding. Covalent reinforcement of relatively fragile protein-protein interfaces may be a useful strategy in minimizing aggregation of non-native structures in multimeric proteins.     

Investigating the Degradation Behaviors of a Therapeutic Monoclonal Antibody Associated with pH and Buffer Species

This study aimed in understanding the degradation behaviors of an IgG 1 subtype therapeutic monoclonal antibody A (mAb-A) associated with pH and buffer species. The information obtained in this study can augment conventional, stability-based screening paradigms by providing the direction necessary for efficient experimental design. Differential scanning calorimetry (DSC) was used for studying conformational stability. Dynamic light scattering (DLS) was utilized to generate B ₂₂*, a modified second virial coefficient for the character of protein-protein interaction. Size-exclusion chromatography (SEC) and hydrophobic interaction chromatography (HIC) were employed to separate degradation products. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used for determining the molecular size and liquid chromatography mass spectrometry (LC-MS) were used for identifying the sequence of the separated fragments. The results showed that both pH and buffer species played the roles in controlling the degradation behaviors of mAb-A, but the pH was more significant. In particular, pH 4.5 induced additional thermal transition peaks occurring at a low temperature compared with pH 6.5. A continual temperature-stress study illustrated that the additional thermal transition peaks related to the least stable structure and a greater fragmentation. Although mAb-A showed the comparable conformational structures and an identical amount of aggregates at time zero between the different types of buffer species at pH 6.5, the aggregation formation rate showed a buffer species-dependent discrepancy over a temperature-stress period. It was found that the levels of aggregations associated with the magnitudes of protein-protein interaction forces.     

Functional aspects of cellular microcompartmentation in the development of neurodegeneration: Mutation induced aberrant protein-protein associations

Research in the last 10 years has revealed that the development of neurodegeneration is a multistep process during which one or few specific mutant protein species of altered conformation initiate aberrant protein-protein interactions resulting in aggregates forming plaques. This review focuses on the heteroassociations of the mutant proteins with subcellular structures, such as cytoskeleton, cell membranes or with glycolytic enzymes, which may be crucial in the initiation of neurodegeneration such as in Huntington's disease or Alzheimer's disease. Triosephosphate isomerase enzymopathy is a unique glycolytic enzyme deficiency coupled with neurodegeneration. We present data on the mutation induced misfolding process, which likely plays a crucial role in the enhanced associations of the enzyme with the truncated fragment of the isomerase, with the red cell membrane or with the microtubular network. On the basis of our recent clinical and experimental results obtained with two compound heterozygote Hungarian brothers it became obvious that the mutations alone are not sufficient to explain the development of the neurological sympthomes. This underscores the fact that the mutations alone are not enough for the development of the clinical phenotype of a disease.     

Ultrastructural organization of recombinant Marburg virus nucleoprotein: comparison with Marburg virus inclusions

HeLa cells expressing the recombinant Marburg virus (MBGV) nucleoprotein (NP) have been studied by immunoelectron microscopy. It was found that MBGV NPs assembled into large aggregates which were in close association with membranes of the rough endoplasmic reticulum. Further analysis of these aggregates revealed that NPs formed tubule-like structures which were arranged in a hexagonal pattern. A similar pattern of preformed nucleocapsids was detected in intracellular inclusions induced by MBGV infection. Our data indicated that MBGV NP is able to form nucleocapsid-like structures in the absence of the authentic viral genome and other nucleocapsid-associated proteins.     

A high-throughput screening method for small-molecule inhibitors of the aberrant mutant SOD1 and dynein complex interaction

Aberrant protein-protein interactions are attractive drug targets in a variety of neurodegenerative diseases due to the common pathology of accumulation of protein aggregates. In amyotrophic lateral sclerosis, mutations in SOD1 cause the formation of aggregates and inclusions that may sequester other proteins and disrupt cellular processes. It has been demonstrated that mutant SOD1, but not wild-type SOD1, interacts with the axonal transport motor dynein and that this interaction contributes to motor neuron cell death, suggesting that disrupting this interaction may be a potential therapeutic target. However, it can be challenging to configure a high-throughput screening (HTS)-compatible assay to detect inhibitors of a protein-protein interaction. Here we describe the development and challenges of an HTS for small-molecule inhibitors of the mutant SOD1-dynein interaction. We demonstrate that the interaction can be formed by coexpressing the A4V mutant SOD1 and dynein intermediate complex in cells and that this interaction can be disrupted by compounds added to the cell lysates. Finally, we show that some of the compounds identified from a pilot screen to inhibit the protein-protein interaction with this method specifically disrupt the interaction between the dynein complex and mtSOD1 but not the dynein complex itself when applied to live cells.     

Combined effect of photoreactivation and temperature on the survival of cells from E. coli B, irradiated with short-wave UV light

A study was made of the survival curves of E. coli B(fil+/lon-) exposed to far UV-light (lambda = 254 nm) plated immediately after liquid holding recovery and after photoreactivation and incubated overnight at 37 degrees and 42 degrees. It was shown that the survival rate was always higher at 42 degrees C irrespective of the modification technique applied. Since the temperature-induced modification was significant after complete elimination of pyrimidine dimers (PD) a conclusion was made that UV-light, in addition to PD, induced just one more type of damages modified by temperature.