Authentic hSAA related with AA amyloidosis: New method of purification, folding and amyloid polymorphism.

The secondary amyloidosis of humans is caused by the formation of hSAA fibrils in different organs and tissues. Until now hSAA was thought to have low amyloidogenicity in vitro and the majority of SAA aggregation experiments were done using murine protein or hSAA non-pathogenic isoforms. In this work a novel purification method for recombinant hSAA was introduced, enabling to obtain monomeric protein capable of amyloid aggregation under physiological conditions. The stability and amyloid aggregation of hSAA have been examined using a wide range of biophysical methods. It was shown that the unfolding of monomeric protein occurs through the formation of molten globule-like intermediate state. Polymorphism of hSAA amyloids was discovered to depend on the solution pH. At pH 8.5, rapid protein aggregation occurs, which leads to the formation of twisted short fibrils. Even a slight decrease of the pH to 7.8 results in delayed aggregation with the formation of long straight amyloids composed of laterally associated protofilaments. Limited proteolysis experiments have shown that full-length hSAA is involved in the formation of intermolecular interactions in both amyloid polymorphs. The results obtained, and the experimental approach used in this study can serve as a basis for further research on the mechanism of authentic hSAA amyloid formation.

Bacteriophage T5 dUTPase: Combination of Common Enzymatic and Novel Functions.

The main function of dUTPases is to regulate the cellular levels of dUTP and dTTP, thereby playing a crucial role in DNA repair mechanisms. Despite the fact that mutant organisms with obliterated dUTPase enzymatic activity remain viable, it is not possible to completely knock out the dut gene due to the lethal consequences of such a mutation for the organism. As a result, it is considered that this class of enzymes performs an additional function that is essential for the organism's survival. In this study, we provide evidence that the dUTPase of bacteriophage T5 fulfills a supplemental function, in addition to its canonical role. We determined the crystal structure of bacteriophage T5 dUTPase with a resolution of 2.0 Å, and we discovered a distinct short loop consisting of six amino acid residues, representing a unique structural feature specific to the T5-like phages dUTPases. The removal of this element did not affect the overall structure of the homotrimer, but it had significant effects on the development of the phage. Furthermore, it was shown that the enzymatic function and the novel function of the bacteriophage T5 dUTPase are unrelated and independent from each other.

First Report of Lysozyme Amyloidosis with p.F21L/T88N Amino Acid Substitutions in a Russian Family.

Lysozyme amyloidosis is caused by an amino acid substitution in the sequence of this protein. In our study, we described a clinical case of lysozyme amyloidosis in a Russian family. In our work, we described in detail the histological changes in tissues that appeared as a result of massive deposition of amyloid aggregates that affected almost all organ systems, with the exception of the central nervous system. We determined the type of amyloidosis and mutations using mass spectrometry. Using mass spectrometry, the protein composition of tissue samples of patient 1 (autopsy material) and patient 2 (biopsy material) with histologically confirmed amyloid deposits were analyzed. Amino acid substitutions p.F21L/T88N in the lysozyme sequence were identified in both sets of samples and confirmed by sequencing of the lysozyme gene of members of this family. We have shown the inheritance of these mutations in the lysozyme gene in members of the described family. For the first time, we discovered a mutation in the first exon p.F21L of the lysozyme gene, which, together with p.T88N amino acid substitution, led to amyloidosis in members of the studied family.

Carbonic anhydrase amyloid fibrils composed of laterally associated protofilaments show reduced cytotoxicity.

Cytotoxicity of amyloid fibrils has been shown to depend on their structure. However, specific features of toxic and non-toxic amyloids remain unclear. Here we focus on the relationship between structural characteristics of the fibrils and their cytotoxicity. Bovine carbonic anhydrase B (BCAB) serves as the object of this study because its amyloids reduce cell viability. Limited proteolysis and mass spectrometry were used to determine BCAB regions forming the core of amyloid fibrils. Four BCAB mutants with substitutions reducing hydrophobicity in the regions important for amyloid formation were obtained to study the kinetics of aggregation, structural features, and cytotoxicity of the amyloids. We demonstrate that fibrils of WT BCAB, L78A, L139A, and M239A variants display a pronounced toxic effect on eukaryotic cells, while I208A mutation significantly reduces the cell-damaging effect of amyloids. The data obtained conclude that cytotoxicity of BCAB fibrils does not depend on their length, secondary structure, and exposure of hydrophobic groups to the solvent. A distinctive feature of the low-toxic I208A fibrils is their specific morphology characterized by the lateral protofilaments association and formation of fibril-ribbons.

Under Conditions of Amyloid Formation Bovine Carbonic Anhydrase B Undergoes Fragmentation by Acid Hydrolysis.

The development of many severe human diseases is associated with the formation of amyloid fibrils. Most of the available information on the process of amyloid formation has been obtained from studies of small proteins and peptides, wherein the features of complex proteins' aggregation remain insufficiently investigated. Our work aimed to research the amyloid aggregation of a large model protein, bovine carbonic anhydrase B (BCAB). It has previously been demonstrated that, when exposed to an acidic pH and elevated temperature, this protein forms amyloid fibrils. Here, we show that, under these conditions and before amyloid formation, BCAB undergoes fragmentation by acid hydrolysis to give free individual peptides and associated peptides. Fragments in associates contain a pronounced secondary structure and act as the main precursor of amyloid fibrils, wherein free peptides adopt mostly unstructured conformation and form predominantly irregular globular aggregates. Reduced acidity decreases the extent of acid hydrolysis, causing BCAB to form amorphous aggregates lacking the thioflavin T binding ß-structure. The presented results provide new information on BCAB amyloid formation and show the importance of protein integrity control when working even in mildly acidic conditions.

Identification of Amyloidogenic Regions in Pseudomonas aeruginosa Ribosomal S1 Protein.

Bacterial S1 protein is a functionally important ribosomal protein. It is a part of the 30S ribosomal subunit and is also able to interact with mRNA and tmRNA. An important feature of the S1 protein family is a strong tendency towards aggregation. To study the amyloidogenic properties of S1, we isolated and purified the recombinant ribosomal S1 protein of Pseudomonas aeruginosa. Using the FoldAmyloid, Waltz, Pasta 2.0, and AGGRESCAN programs, amyloidogenic regions of the protein were predicted, which play a key role in its aggregation. The method of limited proteolysis in combination with high performance liquid chromatography and mass spectrometric analysis of the products, made it possible to identify regions of the S1 protein from P. aeruginosa that are protected from the action of proteinase K, trypsin, and chymotrypsin. Sequences of theoretically predicted and experimentally identified amyloidogenic regions were used to synthesize four peptides, three of which demonstrated the ability to form amyloid-like fibrils, as shown by electron microscopy and fluorescence spectroscopy. The identified amyloidogenic sites can further serve as a basis for the development of new antibacterial peptides against the pathogenic microorganism P. aeruginosa.

The presence of cross-ß-structure as a key determinant of carbonic anhydrase amyloid fibrils cytotoxicity.

In most cases high cytotoxicity is characteristic of aggregates formed during lag phase of amyloid formation, whereas mature fibrils represent the depot of protein molecules incapable of damaging cell membranes. However, new experimental data show that in cases of some proteins the fibrils are the most toxic type of aggregates. Meanwhile, structural characteristics of cytotoxic fibrils and mechanisms of their cell damaging action are insufficiently explored. This work is dedicated to studying amyloid aggregation of bovine carbonic anhydrase (BCA) and effect of aggregates formed at different stages of amyloid formation on viability of the cells. Here we demonstrate that oligomers formed during lag phase do not decrease cell viability, whereas protofibrils and amyloids of BCA are cytotoxic. Obtained results allow concluding that toxicity of BCA aggregates is associated with the presence of amyloid cross-ß-structure, which signature is absorbance peak at low wavenumbers at FTIR spectra (1615-1630 cm-1). Our data suppose that cross-ß-core of ВСА amyloid fibrils is responsible for their cytotoxicity.

sw ApoMb Amyloid Aggregation under Nondenaturing Conditions: The Role of Native Structure Stability.

Investigation of the molecular mechanisms underlying amyloid-related human diseases attracts close attention. These diseases, the number of which currently is above 40, are characterized by formation of peptide or protein aggregates containing a cross-ß structure. Most of the amyloidogenesis mechanisms described so far are based on experimental studies of aggregation of short peptides, intrinsically disordered proteins, or proteins under denaturing conditions, and studies of amyloid aggregate formations by structured globular proteins under conditions close to physiological ones are still in the initial stage. We investigated the effect of amino acid substitutions on propensity of the completely helical protein sperm whale apomyoglobin (sw ApoMb) for amyloid formation from its structured state in the absence of denaturing agents. Stability and aggregation of mutated sw ApoMb were studied using circular dichroism, Fourier transform infrared spectroscopy, x-ray diffraction, native electrophoresis, and electron microscopy techniques. Here, we demonstrate that stability of the protein native state determines both protein aggregation propensity and structural peculiarities of formed aggregates. Specifically, structurally stable mutants show low aggregation propensity and moderately destabilized sw ApoMb variants form amyloids, whereas their strongly destabilized mutants form both amyloids and nonamyloid aggregates.

High affinity anchoring of the decoration protein pb10 onto the bacteriophage T5 capsid.

Bacteriophage capsids constitute icosahedral shells of exceptional stability that protect the viral genome. Many capsids display on their surface decoration proteins whose structure and function remain largely unknown. The decoration protein pb10 of phage T5 binds at the centre of the 120 hexamers formed by the major capsid protein. Here we determined the 3D structure of pb10 and investigated its capsid-binding properties using NMR, SAXS, cryoEM and SPR. Pb10 consists of an α-helical capsid-binding domain and an Ig-like domain exposed to the solvent. It binds to the T5 capsid with a remarkably high affinity and its binding kinetics is characterized by a very slow dissociation rate. We propose that the conformational exchange events observed in the capsid-binding domain enable rearrangements upon binding that contribute to the quasi-irreversibility of the pb10-capsid interaction. Moreover we show that pb10 binding is a highly cooperative process, which favours immediate rebinding of newly dissociated pb10 to the 120 hexamers of the capsid protein. In extreme conditions, pb10 protects the phage from releasing its genome. We conclude that pb10 may function to reinforce the capsid thus favouring phage survival in harsh environments.

Substitutions of Amino Acids with Large Number of Contacts in the Native State Have no Effect on the Rates of Protein Folding.

Various effects of amino acid substitutions on properties of globular proteins have been described in a large number of research papers. Nevertheless, no definite "rule" has been formulated as of yet that could be used by experimentalists to introduce desirable changes in the properties of proteins. Herein we attempt to establish such a "rule". To this end, a hypothesis is proposed on the effects of substitutions of hydrophobic residues with large number of contacts on free energies of different states of a globular protein. The hypothesis states: Substitutions of hydrophobic residues engaged in a large number of residue-residue contacts would not change the folding rate of a protein but could affect its unfolding rate. This hypothesis was verified by both theoretical and experimental analyses, generating a general rule that can facilitate the work of experimentalists on constructing mutant forms of proteins.

Intrinsic Disorder-Based Design of Stabilizing Disulphide Bridge in Gαo Protein.

In this study, we have used an approach that allows us to determine in what region of the polypeptide chain of protein it is required to insert a disulphide bond in order to stabilize it. In our previous paper [Melnik et al., JBSD. 2012] it was proposed that to search for a "weak" site in the protein, it is possible to use programs (for example, PONDR-FIT and IsUnstruct) finding intrinsic disorder protein regions. We suggested that in structured globular proteins, such programs predict not protein regions in the polypeptide chain disordered under native conditions, but "weakened", feebly stabilized ones. Accordingly, an artificial introduction of SS-bridges using mutations in such regions would reliably result in the protein stabilization. We have taken advantage of this approach to stabilize protein Gαo from Drosophila melanogaster. The designed SS-bridge increased by 4 degrees the melting temperature of one domain of protein Gαo.

Updates on the Production of Therapeutic Antibodies Using Human Hybridoma Technique.

Therapeutic antibodies are implicated into the very promising and fast growing area of pharmaceutics. Human hybridoma technology, allowing generation of natural human antibodies in a native form, seems to be the most direct way that require no additional modifications for production of therapeutic antibodies. However, technical difficulties in human hybridoma creation discovered in the 80s of the last century have switched the mainstream therapeutic antibody development into new directions like display and transgenic mice techniques. These approaches have provided remarkable achievements in antibody engineering within last 15 years, but also revealed other limitations. Thus, it is time to turn back to forgotten human hybridoma technology. In this review, we describe new advances in all components of human hybridoma technology and discuss challenges in generating novel therapeutic mABs based on hybridoma technologies.

Independent of their localization in protein the hydrophobic amino acid residues have no effect on the molten globule state of apomyoglobin and the disulfide bond on the surface of apomyoglobin stabilizes this intermediate state.

At present it is unclear which interactions in proteins reveal the presence of intermediate states, their stability and formation rate. In this study, we have investigated the effect of substitutions of hydrophobic amino acid residues in the hydrophobic core of protein and on its surface on a molten globule type intermediate state of apomyoglobin. It has been found that independent of their localization in protein, substitutions of hydrophobic amino acid residues do not affect the stability of the molten globule state of apomyoglobin. It has been shown also that introduction of a disulfide bond on the protein surface can stabilize the molten globule state. However in the case of apomyoglobin, stabilization of the intermediate state leads to relative destabilization of the native state of apomyoglobin. The result obtained allows us not only to conclude which mutations can have an effect on the intermediate state of the molten globule type, but also explains why the introduction of a disulfide bond (which seems to "strengthen" the protein) can result in destabilization of the protein native state of apomyoglobin.

Genomic analysis of Pseudomonas putida phage tf with localized single-strand DNA interruptions.

The complete sequence of the 46,267 bp genome of the lytic bacteriophage tf specific to Pseudomonas putida PpG1 has been determined. The phage genome has two sets of convergently transcribed genes and 186 bp long direct terminal repeats. The overall genomic architecture of the tf phage is similar to that of the previously described Pseudomonas aeruginosa phages PaP3, LUZ24 and phiMR299-2, and 39 out of the 72 products of predicted tf open reading frames have orthologs in these phages. Accordingly, tf was classified as belonging to the LUZ24-like bacteriophage group. However, taking into account very low homology levels between tf DNA and that of the other phages, tf should be considered as an evolutionary divergent member of the group. Two distinguishing features not reported for other members of the group were found in the tf genome. Firstly, a unique end structure--a blunt right end and a 4-nucleotide 3'-protruding left end--was observed. Secondly, 14 single-chain interruptions (nicks) were found in the top strand of the tf DNA. All nicks were mapped within a consensus sequence 5'-TACT/RTGMC-3'. Two nicks were analyzed in detail and were shown to be present in more than 90% of the phage population. Although localized nicks were previously found only in the DNA of T5-like and phiKMV-like phages, it seems increasingly likely that this enigmatic structural feature is common to various other bacteriophages.

Multi-state proteins: approach allowing experimental determination of the formation order of structure elements in the green fluorescent protein.

The most complex problem in studying multi-state protein folding is the determination of the sequence of formation of protein intermediate states. A far more complex issue is to determine at what stages of protein folding its various parts (secondary structure elements) develop. The structure and properties of different intermediate states depend in particular on these parts. An experimental approach, named µ-analysis, which allows understanding the order of formation of structural elements upon folding of a multi-state protein was used in this study. In this approach the same elements of the protein secondary structure are "tested" by substitutions of single hydrophobic amino acids and by incorporation of cysteine bridges. Single substitutions of hydrophobic amino acids contribute to yielding information on the late stages of protein folding while incorporation of ss-bridges allows obtaining data on the initial stages of folding. As a result of such an µ-analysis, we have determined the order of formation of beta-hairpins upon folding of the green fluorescent protein.

SS-Stabilizing Proteins Rationally: Intrinsic Disorder-Based Design of Stabilizing Disulphide Bridges in GFP.

Abstract The most attractive and methodologically convenient way to enhance protein stability is via the introduction of disulphide bond(s). However, the effect of the artificially introduced SS-bond on protein stability is often quite unpredictable. This raises the question of how to choose the protein sites in an intelligent manner, so that the 'fastening' of these sites by the SS-bond(s) would provide maximal protein stability. We hypothesize that the successful design of a stabilizing SS-bond requires finding highly mobile protein regions. Using GFP as an illustrative example, we demonstrate that the knowledge of the peculiarities of the intramolecular hydrophobic interactions, combined with the understanding of the local intrinsic disorder propensities (that can be evaluated by various disorder predictors, e.g., PONDRFIT), is sufficient to find the candidate sites for the introduction of stabilizing SS-bridge(s). In fact, our analysis revealed that the insertion of the engineered SS-bridge between two highly flexible regions of GFP noticeably increased the conformational stability of this protein toward the thermal and chemical unfolding. Therefore, our study represents a novel approach for the rational design of stabilizing disulphide bridges in proteins.

Sequential melting of two hydrophobic clusters within the green fluorescent protein GFP-cycle3.

The analysis of the three-dimensional structure of green fluorescent protein (GFP-cycle3) revealed the presence of two well-defined hydrophobic clusters located on the opposite sides of the GFP ß-can that might contribute to the formation of partially folded intermediate(s) during GFP unfolding. The microcalorimetric analysis of the nonequilibrium melting of GFP-cycle3 and its two mutants, I14A and I161A, revealed that due to the sequential melting of the mentioned hydrophobic clusters, the temperature-induced denaturation of this protein most likely occurs in three stages. The first and second stages involve melting of a smaller hydrophobic cluster formed around the residue I161, whereas a larger hydrophobic cluster (formed around the residues I14) is melted only at the last GFP-cycle3 denaturation step or remains rather structured even in the denatured state.

Identification and characterization of EctR1, a new transcriptional regulator of the ectoine biosynthesis genes in the halotolerant methanotroph Methylomicrobium alcaliphilum 20Z.

Genes encoding key enzymes of the ectoine biosynthesis pathway in the halotolerant obligate methanotroph Methylomicrobium alcaliphilum 20Z have been shown to be organized into an ectABC-ask operon. Transcription of the ect operon is initiated from two promoters, ectAp(1) and ectAp(2) (ectAp(1)p(2)), similar to the sigma(70)-dependent promoters of Escherichia coli. Upstream of the gene cluster, an open reading frame (ectR1) encoding a MarR-like transcriptional regulator was identified. Investigation of the influence of EctR1 on the activity of the ectAp(1)p(2) promoters in wild-type M. alcaliphilum 20Z and ectR1 mutant strains suggested that EctR1 is a negative regulator of the ectABC-ask operon. Purified recombinant EctR1-His(6) specifically binds as a homodimer to the putative -10 motif of the ectAp(1) promoter. The EctR1 binding site contains a pseudopalindromic sequence (TATTTAGT-GT-ACTATATA) composed of 8-bp half-sites separated by 2 bp. Transcription of the ectR1 gene is initiated from a single sigma(70)-like promoter. The location of the EctR1 binding site between the transcriptional and translational start sites of the ectR1 gene suggests that EctR1 may regulate its own expression. The data presented suggest that in Methylomicrobium alcaliphilum 20Z, EctR1-mediated control of the transcription of the ect genes is not the single mechanism for the regulation of ectoine biosynthesis.